Advances in Cancer Research Volume 49

ADVANCESINCANCERRESEARCH VOLUME 49

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ADVANCES IN CANCER RESEARCH Edited by

GEORGE KLElN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

SIDNEY WE INHOUSE Fels Research Institute Temple University Medical School Philadelphia, Pennsylvania

Volume 49- 1987

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

Orlando San Diego New York Austin Boston London Sydney Tokyo Toronto

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87 88 89 YO

9 8 7 6 I, 4 3 2

I

r

CONTENTS

Interaction of Retroviral Oncogenes with the Differentiation Program of Myogenic Cells STEFAN0 ALEMA AND FRANCO TAT^

I. I1. 111. IV. V.

VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myogenic Differentiation ........................................ Properties of Avian Retroviral Oncogenes and Their Products ......... Effects of Retroviral Oncogenes on Myogenesis in Vitro . . . . . . . . . . . . . . Direct and Indirect Mechanisms Involved in the Block of Differentiation ................................................. Conclusions ...................... :............................ References ....................................................

16 22 25

The fos Oncogene INDER M . VERMAAND W. ROBERT GRAHAM I . Introduction .................................................. I1. Biology and Pathology of the FBJ Virus .......................... 111. Characterization of the FBJ Viral Complex ........................ IV . Structure of the fos Gene and Protein ............................ V. Transformation by fos Gene .................................... VI . Protooncogene fos Expression ................................... VII . Transcription of the Protooncogene fos ........................... VIII . Regulation offos Expression .................................... IX . Future Prospects .............................................. References ...................................................

29 30 32 33 37 41 47 48 49 50

Role of the abl Oncogene in Chronic Myelogenous Leukemia ANNE-MARIE MES-MASSON AND OWENN . W I ~ E I . Introduction

................................................... ............................. 111. Molecular Characterization of P210c-nbl............................. IV. Future Directions ............................................... References .................................................... I1. Structure and Expression of abl Genes

53 55 58 63 71

CONTENTS

The Epstein-Barr Virus and the Immune System

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. The Virus . . . . . . . . . . 111. The EBV Receptor . . IV. B Cell .Activation by

v.

.............

............. .................. ..................

Primary EBV Infecti Cellular Immunity during Acute EBV-Induced Infectious Mononucleosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... VII. Chronic Asymptomatic EBV Infection . . . . . . . . . . VIII. Cellular Iiiin~iinityduring Chronic .4symptoniatic EBV Infection . . . . . IX. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75 77 82 84 95

\‘I.

98 105 107 116 116

The Use of Cell Markers in the Study of Human Hematopoietic Neoplasia WENIN H. RASKISD ASD PHILIP J. FIALKOW

I. Introduction . . . . .. 11. Marker Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Lymphoproliferative Disorders . . . . . . . I\‘. ,Myeloproliferati\~eDisorders . . . . . . . . . . . . . . . . . 1’. >farrow Transplantation . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . References . .......................................

160

Multistage Model of Natural Killer Cell-Mediated Cytotoxicity Involving NKCF as Soluble Cytotoxic Mediators BENJAMIS BOSAVIDA ASD SL-SAN C. WR IC HI.

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... ..... 11. Postulated Mechanisms of the NK CMC Reaction . . 111. Multistage Model of the Mechani and Involving NKCF as Soluble Cytotoxic Mediators . ................... IV. NKCF as Soluble Mediators in N ................ 17. Evidence Supporting the Multistage Model in the M N K CMC Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~71. Properties Which Deterniine NK Sensitivity and Resistance . . . . . . . . . VII. Biochemical Characterization of NKCF . . . . . . . . . . . . . . . . . .

VIII. IX. Clinical Implications of the NKCF System . . . . . . . .

X. Remarks . . . . . . . . . . . . .

. . . . .

.........

............... ...............................

169 170 170 171 172 178 180 180 183 184 185

vii

CONTENTS

Shedding of Human Tumor-Associated Antigens in Vitro and

in Vivo

MEENHARD HERLYN.ULRICH RODECK. AND HILARY KOPROWSKI I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Shedding of Tumor-Associated Antigens in Vitro .................... 111. Shedding of Tumor-Associated Antigens in Vioo .................... IV. Shed Tumor-Associated Antigens and Host Response . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

189 190 201 214 215 216

New Classes of Tumor Promoters: Teleocidin. Aplysiatoxin. and Palytoxin HIROTAFUJIKI AND TAKASHI SUGIMURA I. Introduction .................................................. I1. History and Background of Discoveries of Teleocidin. Aplysiatoxin. ................. and Palytoxin ........................... .............................. 111. Teleocidin Tumor Promoters . . . IV. Aplysiatoxin Tumor Promoters ............ ................. V. I n Vitro and in Vioo Effects of Teleocidins and Aplysiatoxin Derivatives .................................................. VI . Mechanism of Tumor Promotion by TPA-Type Tumor Promoters . . . . . VII . Non-TPA-Type Tumor Promoter. Palytoxin ........................ VIII . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...................................................

223 224 228 238 244 251 254 257 258

Anticarcinogenic Action of Protease Inhibitors WALTERTROLL.RAKOMA WIESNER. AND KRYSTYNA FRENKEL I. I1. I11. IV. V. VI .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Plasma Protease Inhibitors in Disease ...................... Inhibition of Carcinogenesis by Protease Inhibitors . . . . . . . . . . . . . . . . . . Contribution of Oxygen Radicals to Carcinogenesis .................. Effect of Protease Inhibitors on Selective DNA and RNA Amplification. . Summary ...................................................... References ....................................................

265 266 268 274 278 279 280

On the Epidemiology of Oral Contraceptives and Disease Ross L. PRENTICE AND DAVIDB . THOMAS

.

I Introduction .................................................. I1. Study Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285 287

viii

CONTENTS

111. Oral Contraceptives and ,Mortality. An Overview . . . . . . . . . . . . . . . . . . . IV . Oral Contraceptives and Disease of the Circulatory System . . . . . . . . . . . V . Oral Contraceptives and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Oral Contraceptives and Other Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Risk-Benefit Summary and Future Research Needs . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

295 300 335 371 374 393

INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

403

INTERACTION OF RETROVIRAL ONCOGENES WITH THE DIFFERENTIATION PROGRAM OF MYOGENIC CELLS Stefan0 Alerna lstituto di Biologia Cellulare, Consiglio Nazionale delle Ricerche, 00196 Rome, Italy

Franco Tat6 Dipartimento di Biologia Cellulare e dello Sviluppo. Sezione Scienze Microbiologiche, UniversitA "La Sapienza." 00185 Rome, Italy

I. Introduction

It is generally agreed that in vitro transformation of differentiating cells is frequently accompanied by the appearance of two remarkable events, namely, an altered control of proliferation and perturbations in the expression of a given cell-specific differentiation program. The loss of growth control after transformation with oncogenic viruses has been studied mostly in fibroblastic cells, i.e., mammalian established cell lines or early passage avian and rodent fibroblasts. These studies have allowed the identification of several properties of transformed cells that distinguish them from their normal counterparts. On the other hand, the relationship between transformation and expression of differentiated traits has remained an elusive and poorly ,understood phenomenon. The reasons for the slow progress in this field can be attributed to the complexity of the regulatory mechanisms underlying tissue-specific gene expression, to the heterogeneity in the coordinate regulation of proliferation and terminal differentiation exhibited by different cell types, and to the existence of more than one mechanism of transformation by the oncogenic agents utilized. Indeed, the formulation of relevant hypotheses and their experimental verification may be facilitated by the use of in vitro differentiating systems of relatively low complexity. Muscle cells are a particularly attractive model, in view also of the considerable background of information available (Pearsons and Epstein, 1982). 1 ADVANCES IN CANCER RESEARCH, VOL. 49

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

STEFAN0 ALEMA AND FRANC0 TAT6

The aim of this article is to review recent data on the interference exerted by various retroviral oncogenes on the expression of the differentiation program of skeletal muscle cells, and, by comparison with the effects exerted on other “simple” systems, to subsume the various experimental observations into a more coherent framework. In the following sections our intention is to emphasize work performed with nonestablished cells using retroviruses as transforming agents, because differentiation of continuous cell lines may not be representative of the normal regulation of differentiated functions and transformation by other agents is not adequately efficient. II. Myogenic Differentiation

Myogenesis certainly provides the best known model for studying the mechanisms governing the transition from a determined to an overtly differentiated state, defined at the molecular level by the activation and expression of cell type-specific gene products and, at the cellular level, by formation of tissue. All the principal features of myogenesis can be faithfully reproduced and studied in vitro (Fig. 1). Commitment of primitive mesenchymal cells to the presumptive myoblast stage (determination) is an event most likely happening in the embryo and still not fully characterized. The subsequent developmentally distinct stages can be readily recognized in essentially pure cultures of myogenic cells, established from avian embryos of various ages. Primary cultures derived from embryonic muscles consist of both cycling precursors and terminally differentiated, postmitotic muscle cells. Presumptive myoblasts are highly replicating cells, do not appreciably express any muscle-specific function or gene, with the possible exception of desmin (Devlin and Emerson, 1978), and their identity is recognized only retrospectively, when they terminally differentiate. Morphological differentiation is characterized by the acquisition of the unique competence to fuse into long, multinucleated syncytia called myotubes (Figs. 1 and 2). It is fundamental to stress here that a definitive withdrawal from the cell cycle, as suggested by Holtzer and co-workers (Stockdale and Holtzer, 1961; Holtzer et al., 1975b), is now recognized as a prerequisite for fusion and expression of muscle-specific genes in both primary cells and mammalian myogenic cell lines (Nadal-Girard, 1978; Turner, 1978). During skeletal muscle differentiation all myogenic precursors are considered equivalent, and it is commonly accepted that they will differentiate into postmitotic muscle cells in response to environmental stimuli, such as low mitogen concentrations (Konisberg, 1977).

RETROVIRAL ONCOGENES AND MYOGENIC DIFFERENTIATION

@ .......

3

1

D ET E R M I N AT10 N

."

2 TERMINAL

'r'

DIFFERENTIATION

3 FUSION

T PA 4

MAT U RAT I ON

5

FIG.1. A schematic representation of developmentally distinct stages of myogenic differentiation and putative steps affected by oncogenes and tumor promoters. 1, Primitive mesenchymal celi; 2, self-renewing presumptive myoblast; 3, postmitotic myoblast; 4, multinucleated myotube; 5, cross-striated myofiber; TPA, tetradecanoylphorbol acetate.

In terms of cell population the regulation of myogenesis is probably more complicated than it appears from the foregoing formalization. So, for instance, a simple inductive model (mitogen concentrationdriven expansion of, or exit from, the postdeteimination compartment of the lineage) (Fig. 1) does not account for the asynchronous time course of terminal muscle differentiation observed both in uiuo and in uitro. Nor does it help to introduce a stochastic dimension to this model, with data derived from experiments with continuous cell lines, which, unlike nonestablished myogenic cells, can be induced to differentiate synchronously (Nadal-Girard, 1978; Linkhart et al., 1981). Recent findings have been interpreted as consistent with the existence in the end phases of the chick myogenic lineage of a self-renewing stem cell that gives rise to a fixed number of committed precursor cells (Robinson et al., 1984; Quinn et al., 1985). The model raises the

4

STEFAN0 A L E a AND FRANC0 TAT6

FIG.2. Fusion and myosin expression in clonal strains of quail myogenic cells transformed by various oncogenes. (A,B)Phase contrast micrographs of t s RSV-transformed cultures grown at 35°C (A)or 41°C (B).(C)Direct immunofluorescence staining of muscle-specific myosin in t s RSV-infected cells grown at 4 1 T : phenotypically normal myotubes show extensive cross-striation. (D) wt RSV-transformed cells grown in differentiation medium at 37°C: atypical organization of myosin in a representative “revertant” myotube. (E, F) Myosin distribution in “revertant” myotubes from w t AEV- (E) and td 10H- (F)transformed cells grown in differentiation medium at 37°C.

possibility that genetic or cytoplasmic regulatory events controlling muscle gene expression in end-stage cells may occur at the stem cell level (Quinn et al., 1985). The irreversible exit from the proliferative state is followed by a several hundredfold increase in the coordinate synthesis of myofila-

RETROVIRAL ONCOGENES AND MYOGENIC DIFFERENTIATION

5

ment proteins such as skeletal muscle actin, myosin, and tropomyosin (Devlin and Emerson, 1978) and in the activity of intracellular enzymes such as creatine phosphokinase (MM-CPK)and glycogen phosphorylase (Lough and Bishoff, 1977; Shainberg et al., 1971). Acetylcholinesterase (AChE) and acetylcholine receptors (AchRs), which later will be used for neuromuscular transmission, are synthesized de novo and inserted at the cell surface (Vogel et al., 1972; Fluck and Strohman, 1973). The available evidence indicates that this activation is based at the molecular level on the accumulation of specific mRNAs, which in turn is due to a transcriptional activation of specific genes (Caravatti et al., 1982; Hastings and Emerson, 1984; Paterson and Eldridge, 1984) (see also Section V,B). The final step in the myogenic pathway is maturation: myotubes containing hundreds of nuclei start to exhibit cross-striations which are due to assembly of myofibrils and then to the building of sarcomeres (Figs. 1 and 2). Finally, after a few days in culture, myotubes become excitable and spontaneously contract. A review by Pearsons and Epstein, (1982) should be consulted for a more detailed account of myogenesis in vitro. 111. Properties of Avian Retroviral Oncogenes and Their Products

Transforming retroviruses represent excellent tools for the study of cell transformation in vitro. They carry in their genome specific sequences, known as oncogenes, that are responsible for tumor formation in vivo and cell transformation in vitro. Each of the dozen different oncogenes so far found in avian retroviruses cause tumors in only a limited set of tissues, while the same oncogenes show a much more relaxed pattern of selectivity in vitro (Graf and Beug, 1978; Weiss et al., 1982). Recent findings imply that the mechanism of transformation by at least some retroviruses depicts with reasonable fidelity one of the steps involved in the conversion of normal cells to tumor cells during natural oncogenesis (Weinberg, 1985; Zarbl et al., 1985). In addition, carefully characterized viral strains are available that permit an efficient and rapid en masse transformation in vitro, thus avoiding selection and cloning of rare transformants. The conditional temperature-sensitive ( t s ) and nonconditional transformation-defective ( t d ) mutants available for many viral oncogenes enable a genetic approach to identifying the relationship between transformation and differentiation, not always feasible with other oncogenic agents. ts mutants appear particularly suited to this aim since they allow unambiguous identification of cellular phenotypes upon shift to the restrictive temperature. This approach can easily discriminate between a fully re-

6

STEFANOALEMAANDFRANCOTAT6

versible altered control of differentiation, solely due to the transformed state, and more complex derangements in the control of specific gene expression that, beside the transforming event, are dependent on unknown cellular or environmental factors. The only caveat in the use of ts mutants is the residual presence of transforming ability at the restrictive temperature that may vary among different mutants as well as among different host cells, and thus generate potentially ambiguous results. Retroviral oncogenes are derived from a restricted set of cellular genes christened protooncogenes (Duesberg, 1985; Bishop, 1985). The transduction of these cellular genes into the viral genome usually results in the loss of one or more viral structural genes and the appearance of deletions and point mutations in the captured genes. Genetic modifications and the acquisition of an autonomous regulatory system provided by the viral enhancer-promoter sequences contained in the long terminal repeats (LTRs) are believed to be the molecular mechanisms required to convert nontransforming protoncogenes into transforming oncogenes (Weiss et al., 1985). Protooncogenes can also be activated to oncogenes while still residing in the cellular genome by such mechanisms as various as point mutation, translocation, enhancer-promoter insertion, and gene amplification (Klein and Klein, 1985). Cellular and viral oncogenes code for specific proteins that can be provisionally allocated into various groups according to their known biochemical properties and their intracellular localization. A summary restricted to avian isolates is shown in Table I (for a recent and comprehensive review, see Bishop, 1985). The largest group includes oncogene products endowed with protein kinase activity that may be further divided into three groups:

1. Tyrosine-specific kinases, such as v-src, v-fps, and v-yes. v-src, the oncogene of Rous sarcoma virus (RSV),encodes a phosphorylated protein of M , 60,000 denoted pp6OV-""that has the intrinsic activity of protein kinase specific for tyrosyl residues (for review, see Weiss et al., 1985).pp60v-srcis myristylated at the NH2 terminal and is localized at the plasma membrane of infected cells and at specialized structures such as adhesion plaques (Weiss et al., 1985). v-fps is the oncogene of the Fujinami sarcoma virus (FSV). In this strain the oncogene is fused to a deleted viral structural gene, the gag gene, and this results in a fusion protein of M , 140,000 known as P140g'g-fps whose biochemical properties and intracellular location are very similar to those of pp60v-src(Moss et al., 1984). 2. Tyrosine-specific kinases homologous to growth factor receptors

RETROVIRAL ONCOGENES AND MYOGENIC DIFFERENTIATION

7

TABLE I PROPERTIES OF AVIANRETROVIFIAL ONCOGENES~ Oncogene

Virus

v-src V-fPS v-ros v-yes

Product

Localization

Function

RSV FSV UR2 Y73

Membranes Membrane/cytoplasm Membranes Membranes

P-tyrosine kinase P-tyrosine kinase P-tyrosine kinase P-tyrosine kinase

v-erbB

AEV

Membranes

v-sea

S13

Membranes

Truncated EGFW P-tyrosine kinase P-tyrosine kinase

v-mil

MH2

Cytoplasm

P-serine kinase

v-erbA

AEV

Nucleus/cytoplasm

Thyroid hormone receptor

v-myc v-myb v-ski v-ets

MC29 AMV SKV E26

Nucleus Nucleus Nucleus Nucleus

DNA binding DNA binding ? ?

" For review, see Weiss et al. (1985).

such as v-erbB. Avian erythroblastosis virus (AEV) strain ES4 carries two separtate oncogenes, v-erbA and v-erbB. Genetic analysis has shown that the main transforming capacity resides in erbB and that erbA alone has no detectable effect (Frykberg et al., 1983; Sealy et al., 1983).v-erbB codes for a glycoprotein of M , 74,000 known as g ~ 7 4 " ' ~ ~ (Hayman and Beug, 1984) that represents a truncated version of the epidermal growth factor receptor (EGFR) (Downward et al., 1984). Like the EGFR it has an intrinsic tyrosine-specific protein kinase activity (Kris et al., 1985),and its localization to the plasma membrane is required for erythroid cell transformation (Beug and Hayman, 1984). 3. Cytoplasmic serine-specific kinases such as v-mil (Moelling et al., 1984). Other groups include nuclear proteins such as v-myc, v-myb, and verbA, which is a mutated form of the thyroid hormone receptor (Sap et al., 1986).v-myc is the characteristic oncogene of various avian myelocytomatosis virus (AMV) strains, whose prototype is the MC29 strain. In the MC29 genome v-myc is fused to a deleted gag gene and thus encodes a fusion protein of M, 110,000 denoted PllOgag-myc.v-myc encoded proteins, with or without fusion to gag peptides, are local-

8

STEFAN0 ALEMA AND FRANC0 TAT6

ized mainly in the nucleus and are able to bind DNA in vitro (Moelling, 1985). From a functional standpoint oncogenes may be classified according to their ability to cooperate in the tumorigenic conversion of early passage rodent fibroblasts. Full transformation of these cells requires the concerted action of two oncogenes possibly belonging to different complementation groups: nuclear or myc-like and cytoplasmic or raslike oncogenes (Land et al., 1983; Ruley, 1983). Avian cells, however, would appear to differ considerably from nonestablished rodent cells, as they are efficiently transformed by single members of either complementation group (see Section IV). IV. Effects of Retroviral Oncogenes on Myogenesis in Vitro

It is almost a decade since it was independently reported (Holtzer et

al., 1975a; Fiszman and Fuchs, 1975) that primary cultures of chick embryo muscle cells could be transformed by RSV and that transformation prevented the formation of multinucleated myotubes. Instrumental for this discovery was the use of ts-mutants of v-src. Thus, at the permissive temperature (35°C) the majority of replicating transformed myogenic cells in standard culture medium fail to (1) withdraw from the cell cycle, (2) initiate the synthesis of muscle-specific products, (3) assemble striated myofibrils, and (4)fuse into multinucleated myotubes. However, when these cells are shifted to the nonpermissive temperature (41"C), many withdraw from the cell cycle, assemble myofibrils, and form multinucleated, spontaneously contracting myotubes (Fig. 2) which express muscle-specific proteins (Fiszman, 1978). It was concluded from these early studies that RSV does not irreversibly cancel, but only suppresses the muscle-specific terminal differentiation program and that this block is under the continuous control of the transforming oncogene. A. SPONTANEOUS DIFFERENTIATION OF RSV-TRANSFORMED MYOBLASTS

The above-mentioned data and the absence of biochemical differentiation at the permissive temperature (Moss et al., 1979) were also interpreted as consistent with the notion of a stable block exerted by v-src on the in vitro differentiation of transformed myoblasts. However, single cell analysis of RSV-transformed myoblasts has shown the unequivocal presence of terminally differentiated, mono- and multinucleated cells in uniformly transformed cultures of ts RSV-infected

RETROVIRAL ONCOGENES AND MYOGENIC DIFFERENTIATION

9

myoblasts (Tat6 et al., 1983).The medium composition was found to play a role in allowing or preventing this spontaneous differentiation. Thus, upon cultivation in growth medium (GM), only a very small percentage (1-2%) of the population expresses a significant level of muscle-specific myosin; in sister cultures cultivated in differentiation medium (DM), a larger fraction (up to 20-30%) can differentiate and fuse into small, atypical myotubes that synthesize muscle-specific myosin, desmin, AChR, and MM-CPK (Tat6 et al., 1983). We shall refer to these myotubes as "revertant myotubes," in order to distinguish them from bona fide myotubes arising from either uninfected cells or ts RSV-transformed cells at the restrictive temperature (Fig. 2). Although revertant myotubes express a muscle-specific gene repertoire qualitatively similar to that of normal cells, they exhibit peculiar characteristics: they often have a flattened, irregular shape with nuclei confined to a centrally located area of the sarcoplasm. Immunofluorescence studies show that myosin is not organized into the musclespecific sarcomeric, striated myofibrils and is rather diffuse or preferentially located in the perinuclear region of the myotube (Fig. 2). It is possible to manipulate the system further, by using dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA), typical inducers of erythroid differentiation (Friend, 1979). These compounds prevent in a reversible manner spontaneous differentiation of wild-type ( w t ) RSV-transformed muscle cells promoted by DM. The inhibitory effect is not observed in uninfected muscle cells and is similar to that exerted by the same compounds on rat myogenic cell lines (Blau and Epstein, 1979). The development of revertant myotubes is also observed in homogeneous cell populations represented by clonal strains of quail myoblasts transformed by w t RSV, selected for anchorage independence, the most stringent criterion for in vitro transformation (Kahn and Shin, 1979).The incidence of spontaneous differentiation in independent clones is variable, ranging from 5 to 50% of nuclei in revertant myotubes after cultivation in DM (Tat6 et al., 1982; Falcone et al., 1985). A comparison between two clones selected as representative of high and low tendency to differentiate indicates that the poorly differentiating clone expresses the RSV provirus better than the highly differentiating one, as suggested by a 3-fold higher level of pp6OV-"" kinase activity and a 6-fold higher production of progeny virus (S. Alema and F. Tat6, unpublished). Albeit limited this observation suggests a correlation between levels of transforming protein and proneness to spontaneous differentiation. While it is clear that t s RSV-transformed myoblasts hardly express

10

STEFANOALEMAANDFRANCOTAT6

biochemical markers of differentiation at the permissive temperature, these cells can still undergo two distinguishable processes of terminal differentiation: (1)upon shift to restrictive temperature the suppression of the transforming activity of pp60v-srcallows the expression of the differentiation program in all the myogenic cells; (2) upon cultivation in DM at the permissive temperature, a minor but sizable fraction of transformed myogenic cells initiates the expression of the program by forming revertant myotubes that do not progress to acquire the functional capacity of muscle fibers. In conclusion, expression of v-src prevents differentiation in muscle cells by blocking the transition from the replicating precursor cell compartment to the terminally differentiated one. This block, however, is unstable and is sensitive to environmental cues; thus a variable proportion of the transformed population may escape this constraint and express its program. The mechanism by which revertant myotubes arise remains largely obscure. The finding that they continue to express high levels of pp6OV-""( S . Alema and F. Tat6, unpublished) rules out the possibility that they originate from cells that either have lost the provirus or selectively do not express the src gene. To a first approximation, spontaneous differentiation might be explained by postulating that srctransformed cells normally experience a transient attenuation of the transformed phenotype. During this postulated brief period of normalcy, myoblasts would withdraw from the cell cycle and commit themselves to terminal differentiation. Since proliferation and differentiation are antithetic processes in muscle cells, a transient event of this sort would be capable of generating a long lasting effect, which is likely to be underscored in other cycling cell types. A possible origin of revertant myotubes from a heterogeneity in the cell population prior to establishment of transformation seems to be ruled out by their presence in both uncloned and cloned transformed cells.

B. ts MUTANTSOF src Avian myogenic cells have been transformed by a variety of different transformation mutants that include ts LA24 (Holtzer et al., 1975a; Falcone et al., 1984), ts NY68 (Fiszman and Fuchs, 1975), t s LA25, ts LA29 (Moss et al., 1979), ts LA30, t s LA32, ts LA33, t s GI201, t s GI251 (Tat6 et al., unpublished), and clonal strains were derived from some of them (Montarras and Fiszman, 1983; Falcone et al., 1985). In spite of scattered reports of residual transformed properties exhibited by some of these mutants at the restrictive temperature in fibroblasts (Wyke and Linial, 1973; Wyke, 1975; Weber and Friis, 1979), genetic

RETROVIRAL ONCOGENES AND MYOGENIC DIFFERENTIATION

11

analysis shows that upon shift to the restrictive temperature the vast majority of infected myoblasts quickly differentiate into myotubes, suggesting a virtually complete return to normality. This was particularly striking in the case of clonal strains that had undergone at least 20-30 generations in vitro and yet were capable of differentiating en masse within a 24-hr shift to 41°C. Cells infected by ts NY68, ts LA24, and ts LA32 have been more thoroughly studied, and subtle as well as macroscopic deviations from the normal maturation pathway have been observed. Two of them relate to abnormalities in the regulation of AChR expression at the surface of infected myotubes. AChRs in myotubes arising from uninfected cells normally aggregate into high density clusters and are down regulated as a consequence of maturation, spontaneous contractile activity and perhaps clustering itself (Changeaux, 1981). The experimental artifice of inactivating sodium channels with tetrodotoxin (TTX) prevents down regulation and results in high levels of receptors (Shainberg et al., 1976). It was shown that (1) t s NY68infected myotubes at 41°C are unable to aggregate AChRs either spontaneously or in the presence of clustering agents (Anthony et al., 1984) and (2) myotubes derived from ts LA24-transformed cells do not down regdate AChRs and are not responsive to TTX treatment (S. Alema and F. Tat6, unpublished). It is tempting to speculate that the abovementioned abnormalities are due to a minimal residual activity of mutated pp60v-srcat the restrictive temperature, undetectable in fibroblasts because of the lack of suitable markers. Alternatively, they might reflect some form of "memory" of the previous transformed state that has not been diluted out because of the shift-induced immediate differentiation and mitotic arrest. In this respect, it has been reported that subtle alterations in the control of the cell cycle traverse are present in ts LA24-infected fibroblasts maintained at the restrictive temperature (Parry et al., 1980). t s LA32 is the only t s transformation mutant of RSV so far identified that induces macroscopic abnormalities in the differentiated phenotype of myotubes at 41°C. The lesion in the src gene of this mutant is representative of a class of alterations in pp6OV-""that does not affect its kinase activity (Stoker et al., 1984).While most mutant pp6Os examined are temperature sensitive for tyrosine phosphorylation both in the test tube and in living cells, pp6OV-""is temperature independent in ts LA32-infected cells. Thus, although morphological transformation is fully thermosensitive, the overall content of phosphotyrosine at 41°C is not decreased as compared to standard mutants such as ts LA29, nor is phosphorylation of p36, the most abundant target protein

12

STEFAN0 A L E d AND FRANC0 TAT6

for pp60v-src(Radke et al., 1980), decreased. Upon shift to 41"C, quail myoblasts transformed by ts LA32 initially differentiate into ostensibly normal myotubes; subsequently, however, instead of proceeding through the maturation phase, these myotubes vacuolate, do not assemble myosin into sarcomeres, and eventually degenerate. A similar picture is observed in revertant myotubes arising at 35°C or when ts RSV-infected myotubes formed at 41°C are shifted back to 35°C and the thermosensitive pp60v-"rcconsequently reactivated (West and Boettiger, 1983) (see also Section V,B). Although the molecular basis of t s LA32 defectiveness has not yet been elucidated, the behavior of ts LASS-transformed myoblasts suggests, on the one hand, that pp60v-srccan interfere with both the initiation and the maintenance of differentiation in muscle cells and, on the other hand, that the same function (defined by the mutation) is responsible for morphological transformation in fibroblasts and block of initiation of differentiation in myoblasts.

c*fPS Results similar to those observed with v-src were obtained with the v-fps oncogene, thus establishing that spontaneous differentiation is not peculiar to v-src but may be a property shared by oncogenes encoding typrosine kinase. Clonal strains of ts FSV-transformed quail myoblasts appear unable to differentiate into myotubes or to express muscle-specific proteins at the permissive temperature. Upon shift to 41"C, the great majority of transformed cells differentiate into phenotypically normal myotubes that show spontaneous contractile activity (Falcone et al., 1985).Like RSV-transformed myoblasts, FSV-infected myoblasts retain the ability to differentiate into revertant myotubes after cultivation in DM, although the incidence is somewhat reduced as compared to src (Falcone et al., 1985).

D. THEerb COMPLEX AEV transformation-specific sequences or erb sequences comprise two different oncogenes: erbB, the main transforming gene in fibroblasts (Sealy et al., 1983) and erythroblasts (Frykberg et al., 1983),and erbA an auxiliary oncogene capable of potentiating the effects of erbB in chick erythroblasts (Frykberg et al., 1983; Kahn et al., 1986). AEVtransformed clonal strains of myoblasts were analyzed and found to be similar to src and fps-transformed myoblasts, as these cells too are prevented from differentiating (Falcone et al., 1985). Upon cultivation

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13

in DM, AEV-transformed cells show the appearance of revertant myotubes that are similar but not identical to those observed in RSVinfected cells. Revertant myotubes in AEV-transformed cells are usually more elongated, contain more nuclei, and show myosin organized in fibrils (Falcone et al., 1985) (Fig. 2C). These findings suggest that the mechanism of transformation by AEV is related to that of other viral tyrosine kinases. Indeed, this contention is supported by the fact the erbB is a truncated form of the EGFR, with intrinsic tyrosine kinase activity (Gilmore et al., 1985). Two different t s mutants of AEV have been employed to transform quail myoblasts, t s 34 and t s 67, both of which bear a mutation in erbB (Graf and Beug, 1983; Choi et al., 1986; P. Scotting, personal communication). t s AEV-transformed clonal strains behave very similarly to wt AEV-transformed myoblasts at the permissive temperature. Surprisingly, upon shift to the restrictive temperature t s AEV-transformed myoblasts do not fully revert to normality and can only generate a modest number of revertant myotubes (Falcone et al., 1985, and unpublished results). There are two possible explanations for the failure of t s AEV-transformed myoblasts to differentiate at the nonpermissive temperature. First, it is known that transformation of fibroblasts by t s AEV is not as stringently temperature dependent as that of t s RSV (Beug and Graf, 1980). This difference may reflect a higher degree of residual transforming activity of the mutated erbB oncogene, perhaps contributed by the potentiating effect of the erbA oncogene. Second, since at least t s 167 bears a single mutation in erbB (Choi et al., 1986), erbA might play a specific role in the transformation and differentiation of myogenic cells, independently from the temperature. Recent data from our laboratories, however, tend to exclude this second possibility. Quail myoblasts were infected with a recombinant retrovirus containing the erbA oncogene alone plus a selectable neomycin-resistance gene (Kahn et al., 1986). Neomycin-resistant, infected cells were fully competent for differentiation into normal myotubes, thus suggesting that erbA alone is neither sufficient to induce transformation nor able to prevent myogenic differentiation. Whether erbA can potentiate the action of src in myoblasts as in adult bone marrow erythroid cells (Kahn et al., 1986) remains to be determined. E. myc Several avian cell types such as adult and yolk sac macrophages (the putative target cells for the induction of myelocytomatosis) (Graf and

14

STEFAN0 ALEMA AND FRANC0 TAT6

Stehelin, 1982; Durban and Boettiger, 1982; Moscovici and Gazzolo, 1982), chondroblasts (AlemB et al., 1985a), and freshly explanted embryonal fibroblasts (Palmieri et al., 1983) are readily transformed in witro by the v-myc oncogene. In the latter cell type v-myc induces focal outgrowth of transformed cells in a dense monolayer as well as anchorage independence for growth (Palmieri et al., 1983). Quail embryo myoblasts are efficiently transformed by infection with MC29 as assessed by colony formation in semisolid media and suppression of differentiation (Falcone et al., 1985). Both uncloned cells and clonal strains of v-myc-transformed myoblasts, however, have lost the option to differentiate into revertant myotubes upon cultivation in DM. This unique property and the paucity of suitable markers to identify replicating myogenic precursor cells raise the question of whether v-myc infection might actually select nonmyogenic cells that are present in primary cultures of muscle cells. Three independent lines of evidence argue against this interpretation. First, ts RSV-transformed clonal strains of myoblasts superinfected by MC29 irreversibly lose the capacity to differentiate either into revertant myotubes at 35°C in DM or into ostensibly normal myotubes at 41°C (Falcone et al., 1985). Second, a complementary approach shows that transformation of muscle cells with v-myc, under conditions that allow replication of nontransformed myoblasts, results in the irreversible suppression of differentiation. Briefly, primary myoblasts were infected by MC29, RSV, and the nontransforming retrovirus RAV-1 and cultivated in the presence of the tumor promoter tetradecanoylphorbol acetate (TPA), which is known to suppress the differentiation of muscle cells and maintain them in an active proliferative state (Cohen et al., 1977; Dlugosz et al., 1983) (see Fig. 1). Upon TPA withdrawal, uninfected and RAV-1-infected cells quickly differentiated, RSV-infected myoblasts generated a small number of revertant myotubes and MC29-infected cells remained mononucleated and replicating (F. Tat6, unpublished). Third, recent data (M. Grossi, personal communication) indicate that MC29-transformed cloned myoblasts synthesize low amounts of desmin, the muscle-specific subunit of intermediate filaments (Bennett et d.,1979; Lazarides, 1980), immunologically detected with a monospecific antibody. It has been reported that several types of cycling myogenic cells, such as quail myoblasts (Devling and Emerson, 1978), TPA-treated chick myoblasts (Dlugosz et al., 1983), and rhabdomyosarcoma cells in viwo (Osborn et al., 1984),express low levels of desmin. It is not yet clear whether desmin expression in those cells is induced by an unknown event that dissociates the expression of this

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15

gene from the expression of the other contractile proteins, or whether it is a specific marker normally expressed in the penultimate compartment of the myogenic lineage.

F. td 10H It is unfortunate, and to some extent surprising, that ts-mutants of the myc oncogene are not yet available. Some recently isolated t s mutants of the MH2 strain of AMV may bear a conditional lesion in myc (von Weizsaecker et al., 1986; Palmieri, 1986), but the presence of a second oncogene, v-mil, precludes a meaningful genetic analysis of v-myc-induced block of differentiation. There are, however, a few well-characterized nonconditional mutants of v-myc; the most studied of these is the td mutant 10H, derived from the MC29 strain. td 10H bears a long deletion (600 bp) in the myc sequence that results in loss of pathogenicity in uiuo (Enrietto et al., 1983). It no longer transforms macrophages in uitro but still transforms fibroblasts (Ramsay et al., 1980). td 10H is therefore referred to as a partial transformation-defective mutant because of the restricted host range for transformation as compared to MC29. td 10H tranforms myoblasts in uitro, but the establishment of transformation follows slower kinetics as compared to MC29. Furthermore, when freshly infected cells are plated in soft agar, they show a 5- to 7day lag before starting to grow, whereas such lag is not observed for parental MC29-infected cells. td 10H-transformed clonal strains retain the ability to differentiate into revertant myotubes that appear to express and accumulate myosin more efficiently than is observed with vsrc (Fig. 2D). Altogether these results confirm the contention of an inherent weakness of the mutated v-myc oncogene and confirm that myoblasts are target cells for v-myc transformation in uitro. G . ski v-ski is the oncogene of the SKV 770 strain of avian carcinoma virus, which was probably generated in uitro by passage of td B77, a transformation-defective mutant of the B77 strain of RSV, in chick embryo fibroblasts (Stavnezer et al., 1981). v-ski is currently classified as a nuclear oncogene on the basis of the intracellular localization of its product P1lOgag-ski-pOz (Barkas et al., 1986).A. E. Barkas et al. (personal communication) have made the unexpected observation that v-ski, beside transforming fibroblasts in uitro, is capable of rescuing the myogenic potential of uncharacterized quail embryo cells (QECs),

16

STEFAN0 ALEMA AND FRANC0 T A T 6

under conditions which are normally suboptimal for in uitro myogenesis. When QECs are infected with SKV virus and subcultivated at clonal density, the majority of clones show the presence of contracting myotubes, whereas uninfected cells give rise to nonmyogenic clones. Myotubes in SKV-infected myogenic clones are eventually overgrown by mononucleated replicating cell which have lost the option to differentiate. Induction of myogenesis is not observed in cultures prepared from defined, non-muscle organs and, therefore, it represents a cell type-specific phenomenon. Interpretation of these data is hindered by the fact that the experimental conditions are not directly comparable to those described in the preceding sections. Although v-ski bears little or no homology with other nuclear oncogenes, one can reasonably consider v-ski as a weak oncogene of the myc type and thus postulate that in the process of transformation by v-ski a proportion of cryptic myogenic cells can express their differentiative program as a consequence of low to intermediate levels of v-ski product accumulation. During this hypothetical process, these cells would terminally differentiate into myotubes, whereas the remaining undifferentiated cells would reach levels of vski expression adequate for full transformation and consequent irreversible loss of the differentiative potential. This speculative argument is given some support by the effects described in td l0H-transformed myoblasts (Section IV,F). An alternative view entails a specific, inductive effect of v-ski on myogenic cell differentiation, which would echo the differentiation-inductive effects of v-src and v-ras on PC12 pheochromocytoma cells (AlemA et al., 198513; Noda et al., 1985; Bar-Sagi and Feramisco, 1985). Given these uncertainties, a careful vis B vis comparison of ski with other nuclear oncogenes under the same experimental conditions is clearly required. V. Direct and Indirect Mechanisms Involved in the Block of Differentiation

The effects of src and other oncogenes on the expression of myogenic differentiation are prima facie in keeping with the notion that transformation negatively affects differentiation. For instance over the past several years a number of authors have described that src specifically suppresses tissue-specific genes or functions such as melanin synthesis in retinal melanoblasts (Boettiger et d., 1977), type I1 collagen synthesis in chondroblasts (Adams et al., 1982), type I collagen synthesis and sensitivity to ascorbate in tendon cells (Schwarz et al., 1978),and neurotransmitter uptake and surface markers in embryonic neuroretina cells (Brakenbury et al., 1984; Casalbore et al., 1987).

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17

In all these systems the block of differentiation is sufficiently stable and thus it has been unequivocally assessed. The phenomenology of the block of differentiation observed in transformed erythroid cells appears to be more complex. Erythroid cells from adult bone marrow are efficiently transformed by erbA plus erbB oncogenes, and the block of differentiation is rather tight, resulting in the presence of an exiguous proportion of hemoglobin-positive (HB+) infected cells (Palmieri et al., 1982). In the same cell type, transformed either by erbB alone or by oncogenes such asfps, src, sea, or ras, the block of differentiation appears to be very unstable, and most of the transformed clones examined have a high content of HB+ cells (Kahn et al., 1984, 1986). In contrast, Jurdic et al. (1985) have shown that, unlike the adult cells, yolk sac erythroblasts transformed by erbA plus erbB show a high degree of spontaneous differentiation. These seemingly discrepant results represent a good example of the heterogeneity of data that bedevils the understanding of the relationship between transformation and differentiation. The main conclusion that can be provisionally drawn is that transformation interferes with the expression of differentiation and that the stability of this interaction may vary according to the cell type and the oncogene analyzed. The presence of spontaneously differentiated cells in a transformed population ought not to be surprising, being a common finding shared by transformed cells in culture and tumors in uiuo. The histological classification and identification of tumors is actually based on the residual expression of tissue-specific differentiated traits. It should be stressed that even in those situations characterized by a high degree of spontaneous terminal differentiation, a sizable proportion of transformed cells maintains unrestrained growth. The question now arises whether the oncogene-induced block of differentiation is relevant to the mechanism of transformation (and tumorigenesis) or is a mere consequence of the loss of proliferation control. With model systems such as those described here this question can now be addressed. We must, however, consider first the different relationship between cell proliferation and terminal differentiation that characterizes different cell types.

A. RELATIONSHIP BETWEEN CELLPROLIFERATION AND DIFFERENTIATION

Terminal differentiation of a cell can be visualized as the transition into the last compartment of its lineage. Lineage compartments are

18

STEFAN0 ALEMA AND FRANC0 TAT6

here defined by discrete cell populations expressing a common and specific repertoire of genes, and hence these cells are to be considered as differentiated (Dienstman and Holtzer, 1975). It so happens that the proliferative potential of terminally differentiated cells can vary widely. For instance, in one extreme example, terminal differentiation of myoblasts is preceded by the irreversible loss of the competence to proliferate, while definitive chondroblasts are still capable of a sustained proliferation rate in vitro (Pacifici et al., 1977). It can therefore be inferred that in myoblasts transformation may affect the control of differentiation either directZy, by interfering with the regulation of muscle-specific gene expression, or indirectly, by preventing the withdrawal from the cell cycle via the alteration of growth control (Fiszman, 1978; Falcone et al., 1984, 1985). On the contrary, transformation may affect differentiation of cycling definitive chondroblasts only if directly causing a derangement in the control of gene expression.

B. src BLOCKSDIFFERENTIATION INDEPENDENTLY FROM CELL PROLIFERATION: DIRECTMECHANISM As mentioned previously, terminal differentiation of myoblasts requires withdrawal from the cell cycle, and, accordingly, this particular process of differentiation is expected to be equally susceptible to either a direct or an indirect mechanism of block. The apparent common block of myogenic differentiation in cells transformed by src,fps, erb, and myc is not surprising in this respect, but it does not provide elements to discriminate between an indirect and a direct mechanism. Two different experimental approaches have been used in an attempt to clarify the point at issue, both using ts RSV-transformed chick myoblasts as valuable reagents. West and Boettiger (1983) have analyzed the selective effect of pp6OV-"" expression in terminally differentiated myotubes by initially allowing the differentiation of ts RSV-transformed myoblasts to occur at the restrictive temperature and subsequently shifting the myotube-containing cultures back to 35°C to reactivate the mutant kinase. A bidimensional gel analysis of contractile proteins showed that reactivation of the transforming protein results in a large reduction of the rate of synthesis of eight skeletal muscle-specific proteins, without significant impairment of the synthesis of a number of housekeeping gene products. Falcone et al. (1984) have exploited mitomycin C (mito-C) as an inhibitor of DNA synthesis and cell replication to study the role of proliferation in srcinduced block of myogenic differentiation. Mito-C-treated ts RSV-

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19

transformed myoblasts do not divide and show a severe reduction in DNA synthesis at the permissive temperature. Arrest of cell proliferation at 35°C was not sufficient per se to induce the expression of the differentiated phenotype; rather, the inactivation of the thermolabile src gene function at 41°C appeared to be essential in order to achieve full differentiation (Falcone et al., 1984). These results strongly suggest that maintenance of a sustained proliferation rate is not sufficient to explain the block of differentiation in src-transformed myoblasts. Furthermore, the data also show that no cell division seems to be required in order to express the terminal differentiation program at 41"C, thus ruling out cell cycle-related commitment as a target for pp60"-5rc. The preceding independent findings corroborate the notion that src can reversibly affect both the initiation of the expression of the myogenic program in replicating myoblasts and the maintenance of the differentiated state in postmitotic myotubes. Together with the suppression of differentiation in replicating definitive cells such as chondroblasts (Pacifici et al., 1977),they constitute circumstantial evidence that v-src affects the expression of the differentiated state via a direct mechanism that is largely independent from the disruption of normal proliferative controls. By the same token, it could be argued that some effects of src on cell multiplication may be secondary to the effects on cell differentiation. For example, fibroblasts and chondroblasts decrease their proliferation rate as they accumulate extracellular matrix in culture; since src prevents the synthesis and accumulation of these products (Adams et al., 1977,1982), src-transformed cells may continue to proliferate after control uninfected cells have stopped. The next question concerning the mechanism of action of v-src on the differentiation of myogenic cells, or other systems, is how, and at what level are changes in gene expression regulated? As mentioned above, several findings lend support to the thesis that one level of action of v-src, leading to a variety of phenotypic consequences, is selective regulation of transcription. The conspicuous number of transcriptional changes elicited by RSV transformation has to be qualified, however, by pointing out that while some of them, such as the activation of a and globin sequences (Groudine and Weintraub, 1984) and other unidentified genes (Groudine and Weintraub, 1980), are shared in the same cell type by other oncogenes such as myc and erb (Groudine and Weintraub, 1980), other changes appear more selective, affecting expression of "luxury" genes. The case of RSV-transformed fibroblasts and chondroblasts is paradigmatic. Expression of

20

STEFAN0 ALEMA AND FRANC0 TAT6

src represses transcription of type I procollagen (Sandmeyer et al., 1981) and fibronectin genes (Tyagi et al., 1983) in chick fibroblasts, whereas it activates the expression of the same genes in chondroblasts concomitantly with suppression of type I1 procollagen (Adams et al., 1982). The remarkably similar effect exerted by src in different specialized cell types suggests that v-src acts at a programmatic level rather than on individual genes, by altering pathways common to the transcription of unlinked lineage-specific genes which are usually coordinately expressed. Gene activation in differentiating muscle cells is an example of such a coordinately regulated process, i.e., all major contractile proteins are synthesized at comparable rates and accumulate in comparable amounts (see Section 11). The susceptibility to transcription of specific genes has been correlated with an "open" chromatin state, as defined, for instance, by sensitivity to nuclease attack (Groudine and Weintraub, 1982), and indeed this has been shown to be the case for muscle-specific genes in myotubes of the L6 myogenic cell line (Carmon et al., 1982). Since ts RSV-transformed muscle cells do not require a round of DNA synthesis in order to differentiate, then the acquisition of changes in chromatin conformation by muscle-specific genes should be independent of DNA synthesis. In addition, mechanisms such as modifications in DNA methylation patterns, also implicated in regulation of gene expression and requiring DNA synthesis for their propagation (Riggs and Jones, 1983), can be excluded as important for this step of differentiation. It can be argued, however, that these changes, perhaps linked to the determination event, have already taken place in srctransformed cells, and thus only need the inactivation of pp6OV-"" to become fully functional. Recent independent results are consistent with a simple model for muscle differentiation, whereby muscle genes are activated by a determination-established,regulatory system that is in turn activated when myoblasts initiate differentiation in response to environmental cues (Konieczny and Emerson, 1985; Blau et al., 1985). Transfection experiments of troponin-I genes into multipotential and muscle lineages of 10T1/2 cells conform to the view that transcription of these genes requires differentiation-specific expression of trans-acting factors, interacting with cis-regulatory elements associated with the contractile protein genes (Konieczny and Emerson, 1985). Interspecies heterokaryons, obtained by fusion of muscle with nonmuscle cells, express muscle-specific proteins encoded by all nonmuscle cell nuclei tested (Blau et al., 1985). These data imply that expression of genes in the nuclei of differentiated cells is susceptible to modulation by tissue-specific trans-acting regulators, in the absence

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21

of DNA replication or cell division. Given the ability of v-src to affect all phases of myogenesis, it may not be unreasonable to speculate that pp60v-srcmay either interact with putative transcription factors (via tyrosine phosphorylation) or interfere with the pathway connecting environmental signals to the regulatory systems. C. myc BLOCKSDIFFERENTIATION VIA UNCONTROLLED CELL PROLIFERATION: INDIRECT MECHANISM

A comparative analysis of the phenotype of different cell types transformed by myc is crucial to understanding the nature of the effects of myc-induced transformation on the expression of a differentiation program. While myc blocks differentiation in myoblasts, myctransformed chondroblasts, although they have acquired an altered growth control, express normal levels of cartilage-specific proteins such as type I1 collagen and type IV proteoglycan (AlemA et al., 1985a). The common inhibition by src and the differential inhibition by myc on the differentiation programs of myoblasts and chondroblasts might find a rational explanation in the fact that chondroblasts, but not myoblasts, can divide while terminally differentiated and by postulating that myc-induced transformation only affects the control of proliferation. Accordingly, myc would indirectly block myogenesis, preventing the required withdrawal from the cell cycle. Such an interpretation is consistent with the available evidence and predicts that the differentiation markers of other replicating cell types should not be affected by myc and that expreimental restoration of growth control in myc-transformed myoblasts should allow a faithful expression of their program. There is increasing evidence that fulfills both of these predictions. (1) v-myc-transformed macrophages are still capable of producing substantial levels of the normal differentiated macrophage products (Graf and Beug, 1978; Durban and Boettinger, 1981). (2) Replicating chick neuroretina cells express specialized functions such as neurotransmitter high-affinity uptake and neural surface markers when uniformly transformed by u-myc, whereas the same functions are virtually abolished by v-src-transformation (Casalbore et al., 1987). ( 3 ) v-myc-transformed clonal strains of myoblasts retain the expression of desmin (see Section IV,E). (4) Upon cocultivation with mammalian fibroblasts such as the C3HlOT112 cell line, the proliferation of myc-transformed quail myoblasts is inhibited, and the myoblasts fuse extensively into small cross-striated myotubes (Tat6 et al., unpublished results). The proposition of a primary effect of v-myc on proliferation finds

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reasonable support in the extensive body of evidence pointing to the implication of the c-myc protooncogene in the regulation of cell growth. Concurrent with induction of cell proliferation c-myc is rapidly induced in quiescent fibroblasts and other cell types by a number of mitogens (Greenberg and Ziff, 1984; Muller et ul., 1984). Regulation of c-myc is effected both at the level of transcription (Greenberg and Ziff, 1984)and by modifications in posttranscriptional RNA stability (Dani et al., 1984; Cole, 1985). Induction of differentiation in a variety of cell lines such as HL60 (Reitsma et al., 1983), embryonal carcinoma cell line F9 (Dony et al., 1985), and mouse erythroleukemia cells (Lachman and Skoultchi, 1984) as well as normal chicken tissues (Gonda et al., 1982) is accompanied by a marked decrease in levels of c-myc mRNA. It was not clear from all these studies, however, whether down regulation of c-myc is causally related to commitment to differentiate or is simply a consequence of the reduced growth rate which accompanies differentiation in these cell types. In a recent study the question of the correlation between c-myc expression and differentiation has been addressed in a rat skeletal muscle cell line (Endo and Nadal-Girard, 1986). It was found that c-myc expression is reduced in biochemically or terminally differentiated cells, although it remains inducible by growth factors. The results would thus indicate that irreversible suppression of c-myc transcription is not required for terminal myogenic differentiation in this cell line and that reexpression of c-myc is not sufficient to affect transcription of muscle-specific genes. According to the “equivalence” viewpoint, one in which there are no essential qualitative differences in the action of c-myc and v-myc, high constitutive levels of expression of the v-myc gene can be envisaged as the driving force for extended proliferation of presumptive myoblasts. Primary rodent fibroblasts often become immortalized if transfected with v-myc or other oncogenes such as ElA, encoding proteins resident in the nucleus and capable of modulating the transcription from “foreign” promoters (Kingston et al., 1985). These functional properties have raised the speculation that the class of nuclear oncogenes may directly induce genes required for cycling and repress other genes required for quiescence and terminal differentiation (Velcich and Ziff, 1984). VI. Conclusions

In this article an attempt has been made to identify common features in the interaction between viral oncogenes and in vitro differentiating cells by comparing the behavior of retrovirus-infected muscle

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23

cells with that of other cell types. Although limited to a few simple cell systems in culture, this comparative analysis has permitted a tentative unifying interpretation of the role of oncogenes in the relationship between acquisition of the transformed state, subversion of proliferation control, and interference with the expression of a differentiation program. This complex and multifarious phenotype may well represent the in uitro counterpart of the pathologist's observations in spontaneous tumors: benign tumors are reasonably well differentiated, whereas malignant ones tend to be anaplastic, with an inverse relationship between the extent of differentiation and the grade of malignancy (Foulds, 1975; Pierce and Wallace, 1971). A prevailing view on the relationship between transformation and differentiation regards transformed cells as "frozen" in a given compartment, incapable of further progression through subsequent compartments of their lineage (Graf and Beug, 1978; Greaves, 1982). The concept is largely derived by the analysis of natural and experimental leukemias, where lineages and compartments within are well defined by specific surface and functional markers. Experimental evidence demonstrates that most leukemias express a near normal panel of specific markers, highly comparable to that of their normal counterparts, the difference lying in the fact that normal cells progress further in the lineage (i.e., the phenotype is transitory) whereas leukemic cells do not (i.e., the phenotype is fixed) (Greaves and Janossy, 1978). In spite of the many differences between leukemic cells and the experimental systems previously discussed, myc-transformed myoblasts can indeed by regarded as frozen in the penultimate compartment of the myogenic lineage. On the other hand, the direct effect exerted by src on the expression of an ongoing differentiation program is patently not compatible with the same conceptual scheme. The common pattern of altered differentiation induced in different cell types by the same oncogene is coherent with a different view of the relationship between transformation and differentiation. To recapitulate, a cytoplasmic oncogene such as src is invariably capable of interfering with the expression of differentiation both in cycling and postmitotic cells belonging to widely different lineages, provided that target cells also become transformed by the oncogene. For example, RSV-infected yolk sac macrophages express high levels of pp6OV-"" (Lipsich et al., 1984), yet do not become transformed and faithfully express their phenotype (Durban and Boettiger, 1981). src-mediated block of differentiation exhibits a variable degree of stability in different cell types and experimental conditions, and thus is compatible with the retention of a residual competence to differentiate. A nuclear oncogene such as myc, on the contrary, reproducibly al-

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ters the proliferation of many avian cell types without ostensibly affecting at least those tissue-specific functions that are compatible with an active proliferation. myc-transformed cells appear to be locked in a continuous proliferative state that may indirectly prevent terminal differentiation, if withdrawal from cell cycle is a prerequisite as is the case of myoblasts. These arguments can be taken further to conclude that the block of differentiation in cells transformed by src is not merely an accident but rather is germane to the mechanism of transformation by src. A last intriguing question is whether the differential effects of myc and src on differentiation have a bearing on the tumorigenic potential of cells transformed by these oncogenes. In the absence of direct evidence, only circumstantial and independent data can be considered. Though avian fibroblasts are efficiently transformed in vitro by both oncogenes (Royer-Pokora et al.,1978), myc-transformed cells do not induce tumors either in histocompatible chickens (Royer-Pokora et al., 1978) or in nude mice (Palmieri et al., 1983), whereas srctransformed fibroblasts are tumorigenic in nude mice (Kahn et al., 1982; Palmieri et al., 1983). These results suggest the possibility that in uitro transformation generates tumorigenic cells only if loss of growth control (i.e., anchorage independence) is accompanied by the suppression of their differentiation potential, as would be expected for src-transformed cells but not for myc-transformed cells. However, peculiar cell properties and/or host factors may contribute to the lack of tumorigenicity of myc-transformed fibroblasts (see Kahn and Shin, 1981), and until the fate of injected cells is clarified this interpretation must be considered as provisional and speculative. Despite this caveat, there is evidence, drawn from experiments with human cell hybrids, compatible with the view that lack of tumorigenicity may be due to the expression or reexpression of the differentiated state of injected cells. Human cell hybrids derived from malignant and normal parental cells express a transformed growth phenotype in uitro, yet are not tumorigenic (Stanbridge and Wilkinson, 1978). Stanbridge and co-workers have carefully analyzed the fate of HeLa x fibroblast and HeLa x keratinocyte stable hybrid cell lines after injection in nude mice (Stanbridge et al., 1982).Nontumorigenic cell hybrids differentiate at the site of injection into a slowly growing normal tissue, whose characteristics appear dictated by the lineage of the normal parent in the hybrid. Rare tumorigenic segregants from both sets of cell hybrids, on the contrary, fail to differentiate and progressively grow up to form tumors (Stanbridge et al., 1982). The aforementioned formalizations and speculations are meant as

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25

heuristic guides to further experimentation. Clearly, other combinations of oncogenes and specialized cell types must be tested before this simplified interpretation of the differential behavior of nuclear and cytoplasmic oncogenes can be validated.

ACKNOWLEDGMENTS We wish to thank our colleagues who contributed critical comments on the manuscript. Work by the authors described in this article was supported by C.N.R. grants under Progetti Finalizzati MPR-SP3, Obiettivo 23, Ingegneria Genetica (86.00085.51), Oncologia (86.00593.44), PS Area 04, and by funds from the AIRC.

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THE fos ONCOGENE lnder M. Verma and W. Robert Graham Molecular Biology and Virology Laboratory, The Salk Institute, San Diego. California 92138

I. Introduction

The realization that a normal cell contains genes capable of inducing neoplasia has opened a new vista in our understanding of the molecular basis of cancer. A decade ago it was observed that the transforming gene of avian sarcoma virus has cellular cognates not only in birds but also in mammals (Stehlin et al., 1976; Spector et al., 1978). Such normal cellular genes are popularly known as oncogenes or protooncogenes by the cognoscenti (Coffin et al., 1981; Bishop, 1983). Nearly two score oncogenes have been identified, and the majority were garnered through the agency of retroviruses, which were first implicated in the formation of tumors at the turn of the century (Hunter, 1984; van Beveren and Verma, 1985). In 1908, Ellerman and Bang used a filtrate from a chicken’s lymphoma to produce lymphomatosis in chickens (Ellerman and Bang, 1908).Peyton Rous used a similar procedure in 1911to isolate a different agent (now known as the Rous sarcoma virus) which produced sarcomas in chickens (Rous, 1911). Over the ensuing years filtrates from tumors of many different animal species were found to contain agents that could induce abnormal growths (Gross, 1951, 1953a,b, 1958; Friend, 1957). One such agent was isolated by Finkel, Biskis, and Jinkin in 1966 from an osteosarcoma that spontaneously arose on the thoracic spine and ribs of a 260-day-old CFUAnl mouse. When an extract made from the osteosarcoma was injected subcutaneously into newborn and 33-day-old mice, they developed bony tumors which arose from the periosteum and grew outward (Finkel et al., 1966). Examination of the tumor tissue by electron microscopy demonstrated viral particles, which were designated as FBJ after its founders (Finkel et al., 1966). Over the next dozen years, much attention was centered on the biology and pathology of the FBJ virus. With the advent of recombinant DNA technology, our laboratory was able to establish the molec29 ADVANCES IN CANCER RESEARCH, VOL 49

Cop)right 0 1987 by Academic P i e s Inc All t~ghtsof reproduction in m y form iesrr\ed

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ular architecture of the fos retroviruses, to identify their transforming gene, v-fos, and to characterize their cellular cognates from mice and humans. In this article we shall attempt to enumerate the biology and pathology of the tumors induced by the fos gene, the structure of the fos gene, and its expression in development, growth, and cellular differentiation. II. Biology and Pathology of the FBJ Virus

The FBJ virus was classified as an osteosarcoma virus (Finkel et al., 1966). However, microscopic examination of FBJ-induced tumors revealed considerable histological variation from region to region within an individual tumor and between tumors. The cellular types included fibroblasts, osteoblasts, osteocytes, and giant cells, and there was a wide variation in the amount of osteoid and degree of ossification. Subsequent pathological studies confirmed the rather wide variation seen in the deposition of osteoid and other interstitial substances (Kellof et al., 1969; Yumoto et al., 1970; Price et al., 1972; Finkel et al., 1972; Ward and Young, 1976). Histological variation in FBJ-induced tumors led Yumoto to subclassify them into osteosarcoma, fibrosarcoma, chondrosarcoma, myxo-fibro-osteosarcoma, osteochondrosarcoma, fibroosteosarcoma, and chondrofibrosarcoma (Yumoto et al., 1970). A common finding among these tumors, however, was the expression of high levels of alkaline phosphatase, a marker for osteoblasts (Price et al., 1972; Ward and Young, 1976). This finding was consistent with the classification of these tumors as osteosarcomas, which express high levels of this enzyme. The production of type I collagen, which is normally produced by osteoblasts and fibroblasts but not chondroblasts, supported the contention that osteoblasts are primary cellular targets for the FBJ virus. Subsequently, it was shown that even those FBJ-induced tumors which arose at sites removed from bone, such as those which arose on the peritoneum, also produced alkaline phosphatase (Ward and Young, 1976). This led to the suggestion that a multipotential cell is the target for the FBJ virus. Infection of a multipotential cell could account for the variable histological appearance of the tumor in that once such a cell is infected, it might still be subject to different hormones or differentiation factors and therefore appear as different cellular types. However, the predominance of tumors around bones, the high levels of alkaline phosphatase expression, the production of type I collagen, and the deposition of osteoid in these tumors all point to osteoblastic precursors as one

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major target for viral infection and transformation. Other cellular types which might be the targets of infection in vivo by the FBJ virus have not been rigorously determined. It is important to emphasize that 90-100% of mice infected with the FBJ virus develop tumors associated with bone (Fig. 1).These tumors often arise on several bones and sometimes the peritoneum indicating multiple sites of viral tumor formation, but metastases are not seen (Ward and Young, 1976). Local complications of tumor growth are responsible for the morbidity and mortality associated with the inoculation of FBJ virus. The tumors grow outward from the periosteum. In contrast, osteosarcomas in humans develop in the deep bony cortex and metastases are prominent. Parosteal sarcomas that arise in humans are more benign and exhibit a growth pattern which is similar to that seen with FBJ-induced tumors in mice (van der Hue1 and von

FIG.1. (A). FBJ-induced tumor. A necropsy on a mouse that had been injected with the FBJ viral complex. The skin has been incised and spread, and the peritoneal cavity has been opened. Two osteosarcomas are evident. One is shown as a large, round, white mass over the sternum, and the other appears as a curved mass involving the right lower ribs. No other tumors are evident. (Courtesy of Dr. T. Curran, Ph.D. thesis.) (B) Histology of an FBJ-induced osteosarcoma. The sternal tumor in A was cut into 1 0 y m sections and stained with hematoxylin and eosin. The normal bone is shown, and there is a mass of the tumor surrounding it. Tumor cell growth is most abundant at the periosteal region. At the periphery, there appears to be invasion of the tumor into the adjacent bone. Notice the disorganized array of tumor cells and the variable amount of interstitial substance in the different regions of the tumor.

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INDER M. VERMA AND W. ROBERT GRAHAM

Ronnen, 1967). Perhaps the FBJ virus would have been better designated as a parosteal tumor virus. Ill. Characterization of the FBJ Viral Complex

Early studies with FBJ viral extracts were hampered by the inability to quantitate the viral titer. Consequently, there was considerable variation in latent periods and incidence of tumors; a low titer extract produced a disease with a long latent period or no disease at all whereas with high titer virus 100% of susceptible mice developed parosteal tumors with latent periods as short as 3 weeks (Finkel et al., 1975). The ability to quantitate the virus came in 1973 when the FBJ viral complex was described (Levy et al., 1973). A helper virus, designated FBJ-MLV, was identified using MLV group-specific antisera and XC plaque assays (Levy et al., 1973). The defective virus, FBJMSV, was identified by focus formation on rat 208F cells. The helper virus produced much higher titers in NIH 3T3 cells, which carry the (Fu-1"") genotype, as compared with BALB/c cells, which carry the (Fv-lbb)genotype, indicating that FBJ-MLV is an N-tropic virus (Levy et al., 1973; Hartley et al., 1970; Weiss et al., 1982). The helper virus (FBJ-MLV) did not induce any disease in mice (Levy et al., 1973). When the entire viral complex (FBJ-MLV and FBJ-MSV) was injected, parosteal tumors developed. In addition, when FBJ-MSV was combined with another helper virus, again parosteal tumors were produced. A titer of at least 1,000 focus-forming units (FFUs) is needed to reliably produce tumors. As opposed to other sarcomas, the FBJ-MSVinduced tumors were found to be transplantable (Levy et al., 1973). It is generally assumed that a tumor which produces virus is not transplantable into an immunocompetent mouse because the viral antigens evoke a vigorous immune response and the tumor is destroyed. The best explanation for these findings is that the FBJ-induced tumors progressed in vie0 such that they were no longer producing virus. The FBJ viral complex was purported to easily establish primary rat or mouse fibroblasts in cultures, whereas other MSVs had been unable to accomplish this (Bather et at., 1968; Levy, 1971; Levy and Rowe, 1971; Levy et al., 1973). Other investigators, however, were unable to efficiently transform and establish rat embryo cells in culture using the FBJ virus (Rhim et at., 1969). Recently another group was unable to establish primary mouse embryo fibroblasts that were transfected with FBJ-MSV DNA, in spite of morphological transformation (Jenuwein et at., 1985).It is doubtful that FBJ-MSV more efficiently transforms or immortalizes cells when compared with other MSVs.

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In addition to the FBJ virus, another retrovirus termed FBR virus, capable of inducing osteosarcomas, was isolated from an osteosarcoma that developed in an X/GF mouse following treatment with gOSr (Finkel et al., 1972). The FBR complex was also shown to contain a defective virus (FBR-MSV) and a B-tropic helper virus (FBR-MLV; Lee et al., 1979). Another retrovirus, RFB complex, which causes benigh osteomas, was described, but we have not been able to grow it (Finkel et al., 1972). Both the FBJ and FBR retroviruses will mediate the formation of primary tumors in mice and induce morphological transformation of cells in culture. By analogy with other defective retroviruses, it was assumed that they harbored oncogenes. The product of FBJ-MSV oncogene was identified to be a 55,000-dalton phosphoprotein (Curran and Teich, 1982b), which was designated v-fos. This 55-kDa protein (p55) was identified by immunoprecipitation using sera from rats that had been injected with F BJ-MSV-transformed cells. Such rats developed tumors, and their sera were referred to as tumor-bearing rat sera (TBRS). This TBRS also precipitated a 39,000-dalton protein of host origin (Curran and Teich, 1982a). p55 could not be precipitated from cells transformed with other oncogenes. v-fos thus represents an essential component in the FBJ viral complex and is apparently responsible for the altered (transformed) morphology of cells infected in tissue culture and for the induction of tumors in uiuo. The FBR-MSV was found to code for a 75,000-dalton gag-fos phosphoprotein that had a region homologous to v-fos (Curran and Verma, 1984; van Beveren et al., 1984; Michiels et al., 1984). Mice injected with the FBR viral complex developed a disease that was identical to that obtained with the FBJ viral complex, i.e., parosteal tumors. As with FBJ-MSV, when FBR-MSV DNA was transfected onto cells they exhibited a transformed morphology. FBR-MSV DNA, however, also established primary cells in culture, and these cells produced tumors when injected into syngeneic or nude mice (Jenuwein et al., 1985). In certain respects then, the FBR viral complex appears to be more potent when compared with the FBJ viral complex. Nevertheless, these findings again placed v-fos sequences in a pivotal role for tumor formation, To gain further insight into the role of the fos gene in the induction of tumors, our laboratory undertook an extensive structural and functional analysis. IV. Structure of the fos Gene and Protein

The complete nucleotide sequences of the FBJ-MSV and FBR-MSV proviral DNAs and the cellular progenitor of the fos gene were deter-

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mined (van Beveren et at., 1983, 1984). Figures 2 and 3 display the organization of the viral and cellular fos genes and their deduced products. The salient features can be summarized as follows: 1. FBJ-MSV proviral DNA contains 4,026 nucleotides, including two long terminal repeats (LTRs) of 617 nucleotides each, 1,639 nucleotides of acquired cellular sequences (v-fos), and a portion of the envelope (enu) gene. 2. Both the initiation and termination codons of the v-fos protein are within the acquired sequences that encode a protein of 381 amino acids, having a molecular weight of 49,601. 3. In cells transformed by FBJ-MSV, a phosphoprotein with an apparent MW of 55,000 (p55) on SDS-polyacrylamide gel electrophoreB'LTR

1

gag

1

FBJ-MLV

1

I

+ATG

TAG-

FBJ-MSV

c-fos (mouse)

FBR-M SV i aa

FIG.2. Organization of the fos gene. The large open box in FBJ-MSV indicates the acquired cellular sequences; solid, vertical bars indicate the initiation and termination codons of v-fos proteins; and the hatched region indicates the carboxyl-terminal 49 amino acids of the v-fos protein encoded in a different reading frame due to deletion of 104 bp of c-fos sequences. The stippled boxes in c-fos (mouse) are the exons; the number of amino acids encoded by each exon is given. The 104-bp sequence that has been deleted in the v-fos sequence is indicated with a box below the line. Unlike the vfos protein, the c-10s protein terminates at a TGA codon. Broken lines indicate the portions of the exon acquired by FBJ- or FBR-MSV from the c-fos gene. Small triangles in FBR-MSV indicate deletion from FBR-MSV as compared with the c-fos gene. The figure is compiled from the data in van Beveren et al. (1983, 1984).

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35

sis (SDS-PAGE) was identified as the transforming protein (Curran et

al., 1982). The discrepancy between the observed size and the size predicted by sequence analysis is likely due to the unusual amino acid composition of the fos protein (10% proline), since the v-fos protein expressed in bacteria has a similar relative mobility (MacConnell and Verma, 1983). 4. The sequences in the c-fos gene that are homologous to those in the v-fos gene are interrupted by four regions of nonhomology, three of which represent bona fide introns. 5. The 104-nucleotide-long fourth region, which is present in both mouse and human c-fos genes, represents sequences that were deleted during the biogenesis of the v-fos gene. (The additional 104 nucleotides in the c-fos gene transcripts do not increase the predicted size of the c-fos proteins, because of a switch to a different reading frame.) There is a 5-bp inverted repeat (GGGCT at position 2555, and AGCCC at position 2656) that overlaps the border of the deletion (van Beveren et al., 1983). It is possible that the 104-bp segment was looped out, either during or after the recombination of the progenitors of FBJ-M SV. 6. The c-fos protein has 380 amino acids, which is remarkably similar to the size of the v-fos protein (381amino acids). 7 . In the first 332 amino acids, the v-fos and mouse c-fos proteins differ at only five residues, whereas the remaining 48 amino acids of the c-fos protein are encoded in a different reading frame from that in the v-fos protein. Thus, the v-fos and c-fos proteins, though largely similar, have different carboxyl termini (Fig. 3A). 8. Despite their different carboxyl termini, both the v-fos and c-fos proteins are located in the nucleus (Curran et al., 1984) (Fig. 4). 9. The mouse and human c-fos genes share greater than 90% sequence homology, differing in only 24 residues out of a total of 380 amino acids (van Straaten et al., 1983). 10. The putative parents of FBJ-MSV (namely, FBJ-MLV and c-fos gene) share a 5-nucleotide sequence at the 5’ end and 10 of 11nucleotides at the 3’ end of the v-fos region. Sequences involved in recombination at the 5‘ end lie in the untranslated region of both FBJ-MLV and mouse c-fos gene. 11. FBR-MSV proviral DNA contains 3,791 nucleotides (specifying a genome of 3,284 bases) and encodes a single gag-fos fusion product of 554 amino acids. 12. Thefos portion of the gene lacks sequences that encode the first 24 and the last 98 amino acids of the 380-amino acid mouse c-fos gene product (Fig. 3A). In addition, the coding region has sustained three

36

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INDER M. VERMA AND W. ROBERT GRAHAM

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THE fOS ONCOGENE

37

small in-frame deletions, one in the p30gag portion and two in the fos region, as compared with sequences of AKR-MLV and the c-fos gene, respectively (van Beveren et al., 1984). 13. The gene product terminates in sequences termedfox (Fig. 2), which are present in normal mouse DNA at loci unrelated to the c-fos gene. The c-fox gene(s) is expressed as an abundant class of polyadenylated RNA in mouse tissue. 14. A retrovirus, FBJ/R, containing amino-terminal sequences of FBJ-MSV and carboxyl-terminal sequences of FBR-MSV containing 268 amino acids was generated, which transformed fibroblasts in v i t r o (Miller et al., 1985). Thus, the gag moiety of FBR-MSV can be removed without affecting its transforming potential. 15. The transformingfos proteins vary in size from 268 amino acids (FBJ/R) to 381 amino acids (FBJ), but they all have nuclear location. Additionally, all fos proteins coimmunoprecipitate a 39K cellular protein whose identify remains obscure. 16. fos-specific antisera were obtained from tumor-bearing rats injected with FBJ-MSV-transformed cells (Curran and Teich, 1982a),as well as from rabbits injected with specific peptides synthesized from various regions of the fos protein (Curran et al., 1985). 17. Both the viral and cellular fos proteins are posttranslationally modified (Curran et al., 1984). The cellularfos protein is more extensively modified with molecular weights ranging from 55,000 to 72,000 (Curran et al., 1984; Kruijer et al., 1984). The extent and precise nature of the fos protein modifications remain unknown, but part of the modifications is due to phosphorylation (J. Barber, personal communication). V. Transformation by fos Gene

Both FBJ-MSV and FBR-MSV containing the fos gene can transform established fibroblast cell lines (Curran et al., 1982; Curran and Verma, 1984). Additionally, it was reported that these viruses induce

differences between c-fos and FBJ protein. I, Position of introns; D, deletion. (B) A schematic comparison p75gug-fos, p55+, and p5P-f"~ proteins. In p75 the gag-encoded portion is indicated with a stippled box, and that encoded by v-fos is shown by the hatched box. The regions of p55*f0s indicated by bold outline boxes and arrows are those portions deleted in ~75gag-f~~. The stippled region in p55"9OS is the carboxylThe numbers refer to the number of terminal portion, which differs from that of ~55c-f~~. amino acids encoded by each region. The data in this figure are compiled from van Beveren et al. (1983, 1984).

THE fOS ONCOGENE

39

foci in primary fibroblasts, myoblasts, and osteoblasts (Jenuwein et al., 1985). Since viral oncogenesis is not the primary mode of induction of human tumors, it is particularly important to study the transforming potential of their cellular cognate, the protooncogene. It was thus intriguing to find that the cellularfos gene can also induce transformation, but requires at least two manipulations: (1)addition of LTR sequences, presumably to increase transcription by producing enhancer sequences, and (2) removal of sequences downstream of the coding domain (Miller et al., 1984). A number of recombinant constructs (Fig. 5) were generated which contained various portions of the viral and cellularfos genes. Briefly, the v-fos and c-fos genes were split into three parts, namely (1) the promoter region and the first 316 amino acids originating from either the v-fos or c-fos gene; (2) the carboxyl terminus, 64 or 65 amino acids of the coding domain of either the v-fos or c-fos gene; and (3) the 3’ noncoding domain [including poly(A) addition signal] originating from either the v-fos or cfos gene. Thus, a construct referred to as VW means that the promoter, the coding domain, and the 3’ noncoding region all originate from the FBJ-MSV proviral DNA, while MMM signifies that complete c-fos (mouse) gene was used. A construct VMV would indicate that the carboxyl terminus of the fos protein is cellular, while M W would contain the viral carboxyl terminus. The results of transformation by various constructs unequivocally demonstrate that both the v-fos and the c-fos proteins can induce cellular transformation. Constructs like VMM which do not efficiently induce transformation are transcribed but are unable to synthesize sufficient p55 fos protein (Miller et al., 1984). In comparison, when the 3’ noncoding sequences are removed as in the transforming construct VM(A),, at least 10 times more fos protein is synthesized (F. Meijlink and T. Curran, unpublished results). It is worth noting that the only difference between transforming W M and nontransforming VMM is the altered carboxyl terminus. Thus it would appear that the noncoding FIG.4. Nuclear localization offos proteins. Indirect immunofluorescence labeling of fos proteins using either tumor-bearing rat antiserum (TBRS) or M2 peptide antiserum (M2).(A) CHOdhfr- cells transfected with pSVdhfr-fos and selected for resistance to 4 x M methotrexate, stained with TBRS. (B) The same cells stained with normal rat serum. (C) 208F rat cells transformed by recombinant clone MMV expressing clfos protein, stained with TBRS. (D) The same field as in C viewed under Nomarski optics. (E) Normal mouse amnion cells stained with TBRS. (F)Same field as in E viewed under Nomarski optics. (G) U-937 cells induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 min and stained with M2 peptide serum. (H) The same field as in G viewed under Nomarski optics.

40

INDER M. VERMA AND W. ROBERT GRAHAM

B

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FIG. 5. Transforming potential of v-fos and c-fos recombinants. (A) Diagrammatic outline of the recombinants. The top line depicts the FBJ-MSV provirus with the v-fos coding region shown by an open box, LTRs, and flanking rat cellular DNA (wavy lines). The middle and lower lines depict the mouse and human c-fos genes, respectively, with coding regions shown as solid boxes, separated by introns. The restriction endonucleases NcoI and SaZI divide the v-fos and mouse cfos genes into three regions and the human c-fos into two regions, as shown. RNA 5' cap and polyadenylation signals are shown. The origins of these plasmids are described (Miller et d., 1984).The arrows in the FBJ-MSV provirus and in the c-fos gene indicate the positions of recombination between the mouse gene and the helper retrovirus that generated FBJ-MSV. Clones were tested for transforming ability by transfection onto rat 208F cells. Plus symbols indicate transformation efficiency of about 200 foci-yg DNA. Minus symbols indicate transforming efficiency of < l o focilyg DNA. (B) Diagrammatic sketch of the c-fos gene. Major conclusions from Fig. 5A are (1)LTR enhancer element is needed, (2) both v-fos and c-fos proteins can transform fibroblasts, and (3) interruption of the sequence in the 3' noncoding domain of c-fos is required for transformation.

sequences interact with the carboxyl terminus to retard transformation. The nature of the sequences in the noncoding domain that is involved in 3' interactions has been extensively analyzed and localized to an A-T-rich 67-bp region located some 500 nucleotides downstream from the end of the coding domain and about 120 nucleotides upstream of the poly(A) addition signal sequence (Meijlink et al., 1985). The precise nature of the 67-bp region in reducing the transforming potential of the c-fos gene is not understood. Two possible mechanisms can be advanced: (1)autoregulation of c-fos protein synthesis by interaction of the c-fus protein with the 67-bp region and/or

THE fOS ONCOGENE

41

the carboxyl terminus or (2)the presence of the 67-bp region influencing the stability of the c-fos mRNA. An underlying basic principle of tumor induction is the assumption that a number of events conspire to acquire the malignant phenotype. Support of this multistep model of carcinogenesis was advanced by the observation that more than one oncogene is required to induce transformation of primary embryo fibroblasts (Land et al., 1983; Ruley, 1983). It is now a general consensus that nuclear oncoproteins like myc, myb, and polyoma large T antigens collaborate with other oncoproteins (cytoplasmic or plasma membrane) to induce transformation of primary embryo fibroblasts (Weinberg, 1985). The fos gene product, despite being a nuclear protein, defies any such categorization because it can induce transformation of primary cultures as well as those of established cell lines (Jenuwein et al., 1985). It appears that FBR-MSV may be more adept at extending self-renewal of nonestablished mouse cells than FBJ-MSV (Jenuwein et al., 1985). The FBR-MSV has undergone several structural alterations as compared to FBJ-MSV, which may in part account for its higher transforming potential. VI. Protooncogene fos Expression

Protooncogenefos is an inducible gene. It is expressed in a wide variety of cell types during development, growth, and differentiation, often in response to a variety of mitogens or differentiation-specific inducers. Table I shows a variety of cell types where fox gene is induced (Verma, 1986). Below we illustrate some specific examples. TABLE I INDUCTION OF c-fos GENE c-fos induction

1. Promonocyte/monomyelocyte

Vit

DB

macrophages

2. pc12 NGF neurites CAMP, K+

3. Partial hepatectomy 4. Spleen cells stimulated with ConA or LPS 5. Resting BALB/c or NIH 3T3 cells stimulated with PDGF, serum, or TPA 6. Hepatocytes stimulated with growth factors 7. A431 + EGF 8. Primary rat pituitary cells + GRF

No c-fos induction 1. Monomyelocyte DMSO granulocytes 2. PC12

chromaffin cells

THE fOS ONCOGENE

43

A. EXPRESSION DURING PRENATAL DEVELOPMENT

During mouse prenatal development, the highest accumulation of c-fos transcripts was detected in late gestational extraembryonal membranes (amnion, yolk sac) (Muller et al., 1982, 1983b; Muller and Verma, 1984; Fig. 6A). Small amounts offos transcript are also detected in placenta and midgestational fetal liver (Muller, et al., 198413). The levels of c-fos transcripts are low during day 10-11 but increase by day 17-18. At day 18 nearly all cells in mouse amnion contain c-fas transcripts as judged by in situ hybridization (Deschamps et al., 1985b). Not only is thefos gene transcribed but also the fos proteins can be identified in day 17-18 amniotic membranes (Fig. 4E; Curran et al., 1984). c-fos expression was also witnessed in human amnion and placenta (Muller et al., 1983a). Expression of c-fos transcripts is generally detected following induction with mitogenic or differentiation-specific agents, but in amnion cells there appears to be a constitutive synthesis of thefos transcripts. It was previously postulated that perhaps the amnion cells are continuously stimulated with growth factors from placenta (Verma et al., 1985). Evidence for this assumption is provided by the experiment that amnion cells in culture do not synthesize c-fos after plating but can be stimulated to make cfos transcripts if dialyzed placental or embryo-conditioned medium is added (Muller et al., 1986). It is not clear, however, if c-fos plays any role during prenatal development. It does not appear to be involved in proliferation of amnion cells because in vitro cultures of amnion cells synthesize DNA without making fos transcripts (Muller et al., 1986).

B. EXPRESSION DURING CELLGROWTH When quiescent mouse fibroblasts are treated with serum or growth factors like PDGF, epidermal growth factor (EGF), or TPA, the protooncogene fos is rapidly induced (Cochran et d., 1984; Greenberg and Ziff, 1984; Kruijer et al., 1984; Muller et al., 1984a; Bravo et al.,

FIG. 6. fos expression. (A) c-fos levels during prenatal development. (1)Placenta, days 7-18; (2) outer (upper) and inner (lower)placenta; (3)extraembryonal membranes including amnion and visceral yolk sac. For details, see Miiller and Verma (1984). (B) cfos transcripts during induction of U-937 cells with TPA. (C) Immunoprecipitation of cfos proteins with M2 peptide sera. For details, see Mitchell et al. (1985). (D) Induction of c-fos mRNA transcripts in quiescent NIH 3T3 cells following addition of PDGF. (E) Immunoprecipitation of c-fos protein with M2 antisera. For details, see Kruijer et al. (1984).

44

INDER M. VERMA AND W. ROBERT GRAHAM

1985; Figs. 6D and 6E). The salient features of these observations can be summarized as follows: 1.Within 2-3 min of stimulation of growth c-fos transcripts can be detected, as measured by hybridization with 32P-labeledcRNA (Kruijer et aE., 1984). Induction of the c-fos gene is at the level of transcription, as measured by nuclear run-off transcription (Greenberg and Ziff, 1984). 2. Maximal levels of induction occur within 20 min (20-fold induction) of the exposure of cells to 0.83 nM purified PDGF. The levels declined by 60 min, and by 240 min few or none c-fos transcripts could be detected. 3. c-fos protein synthesis was maximal with PDGF concentrations that saturate PDGF binding sites at 37°C (1.0 nM) and half-maximal at 0.3-0.5 nM. 4. Addition of cycloheximide resulted in a 50-fold induction. Addition of anisomycin, another inhibitor of protein synthesis, also results in “superinduction” of the c-fos gene (Greenberg et al., 1986). We assume that this “superinduction” represents stabilization of the mRNA since little or no de no00 transcription is observed (Greenberg et al., 1986; Mitchell et al., 1986). 5. We estimate that after 20 min of exposure to PDGF, 0.0001% of NIH 3T3 cell RNA (0.0005% of mRNA) is c-fos mRNA. Assuming a cellular RNA content of 6 pg, this corresponds to about 5-10 copies of fos mRNA per cell. 6. Exposure to PDGF for as little as 30 min induces the synthesis of fos protein which can be detected by immunoprecipitation with fosspecific peptide antisera or TBRS (Kruijer et al., 1984; Muller et al., 1984a). 7 . At least six to eight polypeptides are identified by immune precipitation, most of which represent modified forms of fos protein; however, some non-fos polypeptides are also precipitated. One possibility is that some of them may be related tofos (R-fos) and may react with peptide antisera (Cochran et al., 1984; J. Barber, personal communication). 8. In addition to PDGF or serum, agents like TPA, EGF, and FGF also stimulate transcription of thefos gene (Kruijer et al., 1986; Bravo et al., 1985).

Induction of the c-fos gene when cells transit from the Go to the GI state suggests that it may have a role in the cell cycle. Another protooncogene, c-myc, was shown to be induced during the Go-GI transition (Kelly et al., 1983). The role of protooncogenesfos and myc dur-

THE fOS ONCOGENE

45

ing the cell cycle is difficult to reconcile in view of the results of their invariant amounts during the cell cycle (Thompson et at., 1985; Haan et al., 1985; Bravo et al., 1986). Rapid and transient induction of the fos gene was also observed in regenerating liver 10-60 min following partial hepatectomy (Kruijer et al., 1986). Addition of cycloheximide stabilized the c-fos RNA. Modified forms of the c-fos protein were identified (Kruijer et al., 1986). The in vivo results can be simulated by exposing quiescent adult rat hepatocytes in primary cultures to hepatotrophic factors like EGF or serum. Recently it has been observed that when primary pituitary cells are treated with growth hormone release factor (GRF), c-fos transcripts are rapidly and transiently induced (R. Mitchell, personal communication). The function of the fos protein during cell growth at present remains elusive and largely correlative.

C. EXPRESSION DURING DIFFERENTIATION Protooncogene fos is expressed during the differentiation of a variety of cell lines. Depending on the nature of the inducer, in some cases c-fos expression appears to be lineage specific (Table I). Below we describe some systems. 1 . Hematopoietic Dqferentiation The earliest hint that c-fos expression may be modulated during hematopoiesis came from the work of Gonda and Metcalf (1984).They observed c-fos transcripts when WEH13B murine myeloid leukemia cell line was induced to macrophage-like differentiation with granulocytic colony-stimulating factors. Subsequently it was observed that cfos transcripts can be found in bone marrow and parietal exudates containing macrophages (Muller et al., 1984b, 1985; Mitchell et al., 1985). When human monomyelocytic cell line, HL-60, or promonocytic cell line U-937 are treated with phorbol esters (TPA), the c-fos gene is rapidly induced. Within minutes, the c-fos transcripts can be observed. They accumulate to maximal levels in 30-60 min followed by a decline of 4- to 5-fold, and then the levels remain unchanged for the next 109 hr (Fig. 6B and C; Mitchell et al., 1985). The c-fos protein can, however, be detected for only 60-120 min postinduction. In contrast, when HL-60 cells are induced to differentiate to granulocytes by addition of DMSO, no c-fos transcripts can be observed (Mitchell et al., 1985; Table I). Thus, it is tantalizing to propose some role of c-fos protein during the monocytic differentiation pathway. Recent results from our laboratory, however, show that c-fos expres-

46

INDER M. VERMA AND W. ROBERT GRAHAM

sion is neither sufficient nor obligatory for macrophage differentiation (Mitchell et al., 1986). Two lines of evidence advance these inferences: (1)expression of high levels of c-fos by serum or diacyglycerol (DAG) does not commit U-937 cells to differentiate to macrophages and (2) TPA-resistant HL-60 cell lines can be induced to differentiate to macrophages with 1,25-dihydroxyvitamin D3 without eliciting c-fos expression. At present the role of c-fos during myeloid differentiation remains conjectural and uncertain. 2. Neuronal Differentiation A rat pheochromocytoma cell line (PC12) can be induced to differentiate to neurites upon addition of nerve growth factor (NGF), dibutry1 cyclic AMP, and 50 mM K+. One of the first molecular events to take place upon addition of the differentiation-specific inducers is the rapid but transient expression of the c-fos gene (Curran and Morgan, 1985; Kruijer et al., 1986; Greenberg et al., 1985). Five minutes postinduction, c-fos transcripts can be observed, and they reach maximal levels by 30-60 min. c-fos specific transcripts are no longer detected by 120 min. Modified forms of the c-fos protein are also detected during this period. When PC12 cells are treated with dexamethasone, they differentiate to become chromaffin-like cells. No c-fos expression is observed during this differentiation, again suggesting some lineagespecific expression of the c-fos gene (Kruijer et al., 1986). However, differential neurites upon further addition of fresh NGF showed renewed induction of thefos gene. Thus fos gene expression may be a general anabolic response of the cells to the inducers rather than specific for differentiation. It appears that most inducers of the c-fos gene are also activators of protein kinase C. Thus one could tentatively assume that C-kinase may be involved in c-jos induction. In the absence of any specific inhibitors of C-kinase, it is difficult to undertake definitive experiments, Preliminary experiments suggest that down regulation of C-kinase leads to decreased c-fos induction (R. Mitchell, personal communication). Similarly, the role of the c-fos gene during differentiation remains conjectural and correlative. A definitive role of c-fos protein during differentiation must await experiments where its synthesis is selectively blocked or can be constitutively turned on without addition of inducing agents. 3. c-fos Induced Differentiation Introduction of the c-fos gene into undifferentiated F9 teratocarcinoma cell line leads to endoderm-like differentiation. When a nor-

THE fOS ONCOGENE

47

ma1 c-fos gene or a c-fos gene linked to a metallothionein promoter was introduced by DNA transfection into F9 stem cells, colonies of morphologically altered cells were obtained (Muller and Wagner, 1984). However, the c-fos-induced differentiation was different from chemically induced differentiation because only a part of the expected morphological and biochemical changes were registered. Furthermore, many cell clones were isolated which showed high levels of expression of the c-fos protein without any morphological or biochemical alterations (Ruther et al., 1985). It would thus appear that expression of exogenously added fos gene may not be sufficient to induce complete differentiation of F9 stem cells. Other genes or factors may be required. VII. Transcription of the Protooncogene fos

The rapid and transient induction of c-fos gene transcription lends itself to a search for transcriptional enhancers and inducible sequences in the c-fos gene. Several investigators have recently reported the identification and location of transcriptional enhancer elements of the c-fos promoter (Treisman, 1985; Deschamps et al., 1985a,b; Renz et al., 1985). An element essential for transcriptional activation and inducibility in response to serum is located between nucleotides -276 and -332, relative to the 5’ cap site. When this sequence is linked to a heterologous promoter, however, the extent of induction with either serum or TPA is only 3- to 5-fold (Treisman, 1985; Deschamps et al., 1985a). Furthermore, the transcripts are more stable with increased constitutive levels (Treisman, 1985). By making fusion genes between human c-fos and mouse p-globin genes it has been shown that in addition to the 5’ activating element, transient accumulation of c-fos RNA following induction with serum also requires sequences at the 3’ end of the c-fos gene. The precise nature of these sequences remains unknown, but they are located downstream of the 3’ coding domain and include the interacting 67-nucleotide sequence discussed above. Two points are worth noting: (1) The 5’ upstream sequences essential, for transcriptional activation are conserved between human and mouse c-fos genes; these sequences also contain one of the two DNase I hypersensitive sites (Deschamps et al., 198513).An additional DNase I hypersensitive site is found intragenically in the c-fos mouse gene but not in the human (Renz et al., 1985). (2) The large variety of cell types where fos gene expression can be induced show that, as expected, the fos enhancer is not tissue specific (Deschamps et al., 1985a).

48

INDER M. VERMA AND W. ROBERT GRAHAM

Recently, several investigators have identified inducible factors which specifically bind to the enhancer elements of the c-fos gene (Treisman, 1986; Prywes and Roeder, 1986; Gilman et al., 1986). It appears that the fos-enhancer region has multiple sites of binding to the serum or TPA-inducible transcriptional factors. Surprisingly, induction of c-fos transcription is not inhibited by addition of protein synthesis inhibitors such as cycloheximide, suggesting that synthesis of no new transcriptional factors is required. We hypothesize that transcription of the c-fos gene is modulated by negatively and positively acting cellular factors (Sassone-Corsi and Verma, 1987). Inducible agents such as TPA or serum merely modify the transcriptional factors to allow transcription of the c-fos gene. VIII. Regulation of fos Expression

The fos gene is versatile; the gene product may play a role during development, cellular differentiation, and cell growth. Since the c-fos protein can induce transformation of at least fibroblasts in vitro, it is puzzling that cells expressingfos genes in response to inducers do not succumb to transformation. Perhaps fibroblasts and other cells susceptible to transformation by c-fos protein are not transformed because the expression of the fos protein is transient. It is possible that some cell types, such as peritoneal macrophages or macrophages in culture, are refractory to transformation by fos proteins. In addition to transcriptional activation, the synthesis of the fos gene product may be regulated posttranscriptionally or, even more likely, at the translational level. As mentioned before, c-fos can induce cellular transformation if an A-T-rich stretch of 67 base pairs located downstream of the termination codon is removed. We have no firm idea of the manner in which the 67-base pair sequence influences the synthesis of the cfos protein, but it could either affect the stability of the mRNA or alter the translational efficiency of the c-fos mRNA. Little or no c-fos protein is detected in cells transfected withfos recombinant DNA constructs containing the carboxyl terminus of the c-fos protein and the 67 base pairs. Promonocytic or monomyelocytic cell lines induced to differentiate to macrophages continue to expressfos mRNA for at least 10 days, but thefos protein is detected only for up to 120 min following induction (Fig. 6; Mitchell et al., 1985). It is possible that the fos antiserum is unable to detect fos proteins because they are extensively modified. It is also difficult to comprehend how a protein found exclusively in the nucleus can influence translation of its message. Apparently post-

THE fOS ONCOGENE

49

transcriptional or translational control of the expression of fos gene product is abrogated in mouse amnion cells where both the RNA and protein can be detected during prenatal development. It is possible that, owing to the presence of growth factors in the placenta, the c-fos gene is continuously stimulated in amnion cells. Regardless of the molecular mechanism influencing fos expression, we believe that the natural expression of the c-fos protein does not transform cells because it is synthesized only transiently. In contrast, the v-fos gene escapes this regulation, because of an altered carboxyl terminus, and its sustained synthesis leads to cellular transformation. IX. Future Prospects

In the past 5 years much has been learned about the molecular structure and expression of the fos oncogene. This knowledge, however, has been accompanied by relatively little progress in ascertaining the function of thefos protein in normal cells. Thus delineation of the function of the fos gene product will command the top priority of investigators. Characterization of the nature of the posttranslational modification of the fos protein will be vigorously pursued. Elucidation of the molecular mechanism by which noncoding sequences influence the transforming potential of the c-fos gene remains a formidable challenge. Transcription of the protooncogene fos has elicited considerable interest, and progress is anticipated in defining specific transcriptional factors involved in its induction. The issue of the stability of the c-fos mRNA will be explored in depth because it may offer clues to the mechanism of transient expression in many rapidly inducible genes. Finally, attempts will be made to understand why the fos protein which can be induced in a wide variety of cell types causes only bone tumors. We will perhaps witness the use of tansgenic mice to determine if inappropriate or overexpression of the fos gene product can induce tumors in a wide variety of tissues. Are rearrangements or chromosomal translocation of the fos gene involved in any human tumors? We feel that exquisite regulation of the protooncogene fos offers a model system to understand how the cell treads the fine line between the need for an essential function and the potential of disaster. ACKNOWLEDGMENTS We thank our many colleagues for providing unpublished data and Carolyn Goller for typing the manuscript. We extend special appreciation to Marguerite Vogt for her advice and interest.

50

INDER M. VERMA AND W. ROBERT GRAHAM

This work was supported by grants from the National Institutes of Health and the American Cancer Society to I.M.V. and a grant from the National Cancer Institute to W.R.G.

REFERENCES Bather, R., Leonard, A., and Yang, J. (1968).J. Natl. Cancer Inst. 40, 551-560. Bittner, J. J. (1942). Science 45, 462-463. Bravo, R., Burckhardt, J., Curran, T., and Muller, R. (1985). EMBO J . 4, 1193-1198. Bravo, R., Burckhardt, J., Curran, T., and Miiller, R. (1986). EMBOJ. 5, in press. Cochran, B. M., Zullu, J., Verma, I. M., and Stiles, C. D. (1984). Science 226, 10-80. Coffin, J. M., Varmus, H. E., Bishop, F. M., Essex, M., Hardy, W. D., Martin, G. S., Rosenberg, N. E., Scolnick, E. M., Weinberg, R. A., and Vogt, P. K. (1981).J.Virol. 40,953-957. Curran, T. (1982). Ph.D. thesis, Imperial Cancer Research Fund Labs., London. Curran, T., and Morgan, J. P. (1985). Science 229, 1265-1268. Curran, T., and Teich, N. M. (1982a). Virology 116,221-235. Curran, T., and Teich, N. M. (1982b).J. Virol. 42,114-122. Curran, T., and Verma, I. M. (1984). Virology 135,218-228. Curran, T., Peters, G., Van Beveren, C., Teich, N., and Verma, I. M. (1982).J.Virol. 44, 674-682. Curran, T., Miller, A. D., Zokas, L., and Verma, I. M. (1984). Cell 36,259-268. Curran, T., Van Beveren, C., Ling, N., and Verma, I. M. (1985). Mol. Cell. Biol. 5,107112. Deschamps, J., Meijlink, F., and Verma, I. M. (1985a). Science 230, 1174-1177. Deschamps, J., Mitchell, R. L., Meijlink, F., Kruijer, W., Schubert, D., and Verma, I. M. (1985b). Cold Spring Harbor Symp. Quant. Biol. 50,733-745. Ellerman, V., and Bang, 0. (1908). Zentralb F , Bact. Abt. 146, 595-609. Finkel, M. P., Biskis, B. O., and Jinkins, P. B. (1966). Science 151,698-701. Finkel, M. P., Reilly, C. A., Biskis, B. O., and Greco, J. L. (1972). Proc. Symp. Colson Res. Soc., 24th, pp. 353-366. Finkel, M. P., Reilly, C. A., and Biskis, B. 0. (1975).Front. Radiat. Ther. Oncol. 10,2939. Friend, C. (1957).J . E x p . Med. 105, 304-318. Gilman, M. Z., Wilson, R. N., and Weinberg, R. A. (1986). Mol. Cell. Biol. 6,4305-4316. Gonda, T. J., and Metcalf, D. (1984). Nature (London)310, 249-251. Greenberg, M. E., and Ziff, E. B. (1984). Nature (London) 311,433-438. Greenberg, M. E., Greene, L. A., and Ziff, E. B. (1985). J . B i d . Chem. 260, 1410114110. Greenberg, M. E., Hermanowski, A. L., and Ziff, E. B. (1986). Mol. Cell. Biol. 6,10501057. Gross, L. (1951). Proc. SOC. E x p . Biol. 76, 27-32. Gross, L. (1953a). Proc. SOC. E x p . Biol. Med. 83,414-421. Gross, L. (1953b). Cancer 6, 948-957. Gross, L. (1958). Acta Haematol. 19, 353-361. Haan, S. R., Thompson, C. B., and Eisenmann, R. (1985). Nature (London)314, 369371. Hartley, J. W., Rowe, W. P., and Huebner, R. J. (1970).J . Virol. 5,221-225. Hunter, T. (1984). Sci. Am. 251,70-79.

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Jenuwein, T., Miiller, D., Curran, T., and Miiller, R. (1985). Cell 41,629-637. Kellof, G. S., Lane, W. T., Turner, A. C., and Huebner, R. J. (1969). Nature (London) 223,1379-1380. Kelly, K., Cochran, B. H., Stiles, D., and Leder, P. (1983). Cell 35,603-610. Kruijer, W., Cooper, J. A., Hunter, T., and Verma, I. M. (1984). Nature (London)312, 711-716. Kruijer, W., Skelly, H., Botteri, F., v.d. Putten, H., Barber, J., Verma, I. M., and Leffert, H. (1986).J. Biol. Chem. 261, 7929-7933. Land, H., Parada, L. F., and Weinberg, R. A. (1983). Nature (London)304,596-598. Lee, C. K., Chan, E. W., Reilly, C. A,, Pahnke, V. A., Rockus, G., and Finkel, M. P. (1979). Proc. SOC. E x p . Biol. Med. 162, 214-220. Levy, J. A. (1971).J. Natl. Cancer Inst. 46, 1001-1007. Levy, J. A., and Rowe, W. P. (1971). Virology 45, 844-847. Levy, J. A., Hartley, J. W., Rowe, W. P., and Huebner, R. J. (1973).J. Natl. Cancer Inst. 51,525-539. MacConnell, W. P., and Verma, I. M. (1983). Virology 131, 367-372. Meijlink, F., Curran, T., Miller, A. D., and Verma, I. M. (1985). Proc. Natl. Acad. Sci. U.S.A. 82, 4987-4991. Michiels, L., Pedersen, S., and Merregaert, J. (1984). Int. J. Cancer 33, 511-517. Miller, A. D., Curran, T., and Verma, I. M. (1984). Cell 36, 51-60. Miller, A. D., Verma, I. M., and Curran, T. (1985).J. Virol. 55, 521-526. Mitchell, R. L., Zokas, L., Schreiber, R. D., and Verma, I. M. (1985). Cell 40,209-217. Mitchell, R. L., Henning-Chubb, C., Huberman, E., and Verma, I. M. (1986). Cell 45, 497-504. Muller, R., and Verma, I. M. (1984). Curr. Top. Microbiol. Immunol. 112, 73-115. Miiller, R., and Wagner, E. F. (1984). Nature (London) 311,438-442. Miiller, R., Slamon, D. J., Tremblay, J. M., Cline, M. J., and Verma, I. M. (1982).Nature (London) 299,640-644. Miiller, R., Tremblay, J. M., Adamson, E. D., and Verma, I. M. (1983a).Nature (London) 304,454-456. Muller, R., Slamon, D. J., Adamson, E. D., Tremblay, J. M., Muller, D. J., Cline, M. T., and Verma, I. M. (1983b). Mol. Cell. Biol. 8, 1062-1069. Muller, R., Bravo, R., Burckhardt, J., and Curran, T. (1984a).Nature (London)312,716720. Miiller, R., Muller, D., and Guilbert, L. (1984b). EMBOJ. 3, 1887-1890. Muller, R., Curran, T., Miiller, D., and Guilbert, L. (1985). Nature (London)314, 546548. Muller, R., Miiller, D., Verrier, B., Bravo, R., and Herbst, H. (1986). EMBO J. 5, 311316. Price, C. H. G., Moore, M., and Jones, D. B. (1972). Br. J. Cancer 26, 15-27. Prywes, R., and Roeder, R. G. (1986). Cell 47,777-784. Renz, M., Neuberg, M., Kurz, C., Bravo, R., and Muller, R. (1985). EMBOJ. 4,37113716. Rhim, J. S., Huebner, R. J., Lane, W. J., Turner, A. C., and Rabstein, L. (1969).Proc. Soc. E x p . Biol. Med. 132, 1091-1098. Rous, P. (1911).J. Am. Med. Assoc. 56, 198. Ruley, H. E. (1983). Nature (London)304,602-606. Ruther, U., Wagner, E. F., and Muller, R. (1985). EMBO J. 4, 1775-1781. Sassone-Corsi, P., and Verma, I. M. (1987). Nature (London)326, 507-510. Spector, D. H., Baker, B., Varmus, H. E., and Bishop, J. M. (1978). Cell 13,381-386.

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Stehlin, D., Varmus, H. E., Bishop, J. M., and Vogt, P. (1976). Nature (London) 260, 170- 173. Thompson, C. B., Challoner, P. B., Neiman, P. E., and Groudine, M. (1985).Nature (London) 314,363-366. Treisman, R. (1985). Cell 42, 889-902. Treisman, R. (1986). Cell 46, 567-574. van Beveren, C., and Verma, I. M. (1985). Curr. Top. Microbiol. Zmmunol. 123,73-98. van Beveren, C., van Straaten, F., Curran, T. Muller, R., and Verma, I. M. (1983). Cell 32,1241-1255. van Beveren, C . ,Enami, S., Curran, T., and Verma, I. M. (1984). Virology 135,229-243. van der Heul, R. O., and von Ronnen, J. R. (1967). Boneloint Surg. 99-A 415-439. van Straaten, F., Muller, R., Curran, T., Van Beveren, C., and Verma, I. M. (1983). Proc. Natl. Acad. Sci. U.S.A.80,3183-3187. Verma, I. M. (1986). Trends Genet. 2,93-96. Verma, I. M., Mitchell, R. L., Kruijer, W., van Beveren, C., Zokas, L., Hunter, T., and Cooper, J. A. (1985). In “Cancer Cells 3/Growth Factors and Transformation,” pp. 275-287. Cold Spring Harbor, New York. Ward, J. M., and Young D. M. (1976). Cancer Res. 36,3985-3992. Weinberg, R. A. (1985). Science 230, 770-776. Weiss, R., Teich, N., Varmus, H., and Coffin, J. (1982). “Molecular Biology of Tumor Viruses,” 2nd Ed., pp. 69-75. Yumoto, T., Poel, W. E., Kodama, T., and Dmochowski, L. (1970). Tex. Rep. Biol. Med. 28,145-165.

ROLE OF THE abl ONCOGENE IN CHRONIC MYELOGENOUS LEUKEMIA Anne-Marie Mes-Masson* and Owen N. Witte Department of Microbiology and Molecular Biology Institute, University of California, Los Angeles, California 90024

I. Introduction

Recent developments have provided a wealth of evidence implicating the c-abl oncogene in the human disease chronic myelogenous leukemia (CML).This human leukemia is associated with a consistent chromosomal abnormality, the Philadelphia chromosome (Ph'), which is correlated with the presence of an altered abl mRNA and protein. The altered abl protein in CML shares many features in common with the altered abl protein involved in transformation mediated by the Abelson murine leukemia virus. The structure and function of the different forms of the abl protein are discussed in this article. Particular emphasis is given to the molecular organization of the altered c-abl sequences and their involvement in CML. The clinical and cytogenetic aspects of CML will be only briefly discussed as these topics have been extensively covered in recent reviews (Champlin and Golde, 1986; Sandberg et al., 1986). A. ABELSONMURINELEUKEMIA VIRUS

Abelson murine leukemia virus (A-MuLV)was initially isolated by Abelson and Rabstein (1970) after innoculation of a prednisolonetreated BALB/c mouse with Moloney murine leukemia virus (MMuLV). While M-MuLV usually induces a T cell leukemia, one mouse developed an acute B cell leukemia. The virus isolated from this mouse proved to be a replication-defective, highly oncogenic retrovirus able to transform murine fibroblast and lymphoid cells in vitro (Scher and Siegler, 1975; Rosenberg et al., 1975).The viral RNA * Present address: Institut de Recherche en Biotechnologie, 6100 Royalmount, Montreal, Quebec, Canada H4P 2R2. 53 ADVANCES IN CANCER RESEARCH, VOL. 49

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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was shown to encode a single polypeptide which in the prototype strain of A-MuLV is a phosphoprotein of 160,000 daltons (P160v-Qbl) composed of N-terminal gag polyprotein sequences fused to non-MMuLV sequences (Witte et al., 1978; Reynolds et al., 1978; Shields et al., 1979; Van de Ven et al., 1979). The non-M-MuLV sequences of AMuLV are derived from a host cellular gene, c-abl (Goff et al., 1980; Shields et al., 1979). The c-abl gene is a single copy gene which has been localized to chromosome 2 in mice and chromosome 9 in humans (Goff et al., 1982; Heisterkamp et al., 1982). Antisera directed against the non-M-MuLV protein portion cross-react with a normal in murine cell protein (Witte et al., 1979) of 150,000 daltons (P150c-abl) cells and 145,000 daltons (P145c-abz) in human cells (Konopka et al., 1984a). Much evidence demonstrates that the transforming activity of the vabl gene is localized to the c-abl-derived sequences (Witte et al., 1980a; Reynolds et al., 1980; Rosenberg et al., 1980; Srinivasan et al., 1982; Watanabe and Witte, 1983; Prywes et al., 1983). The c-abl sequences in P160v-Qbz endow the protein with a tyrosine-specific protein kinase activity, an activity initially detected by an autophosphorylation reaction (Witte et al., 1980a,b; Van de Ven et al., 1980; Wang et al., 1982; Konopka et al., 1984a; Foulkes et al., 1985). This activity appears to be essential for transformation since mutants lacking or decreased in tyrosine kinase activity are also transformation defective (Witte et al., 1980a; Rosenberg et al., 1980; Prywes et al., 1985). Transformation by A-MuLV is accompanied by increased levels in total cellular phosphotyrosine, indicating that v-abl functions as a tyrosine kinase in vivo (Sefton et al., 1981, 1983). Moreover, it has been shown that the v-abl protein itself is phosphorylated at tyrosine residues in vivo (Ponticelli et al., 1982; Sefton et al., 1981). Taken together, these results suggest that an altered tyrosine kinase activity of the abl gene in A-MuLV is responsible for its oncogenic potential.

B. CHRONIC MYELOGENOUS LEUKEMIA Chronic myelogenous leukemia (CML) is a human disease resulting from the neoplastic transformation of a pluripotent hematopoietic stem cell (reviewed in Koeffler and Golde, 1981; Champlin and Golde, 1985). This leukemia is associated with a consistent chromosomal abnormality known as the Philadelphia (Ph') chromosome which occurs in over 90% of CML patients (Nowell and Hungerford, 1960; Rowley, 1975). The Ph' chromosome originates from the reciprocal and balanced translocation between chromosomes 9 and 22; the translocation breakpoint has been extensively mapped to

abl

IN CHRONIC MYELOGENOUS LEUKEMIA

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t(9;22)(q34.l;qll.l)(Rowley, 1975; Prakash and Yunis, 1984). The onset of CML is characterized by a relatively benign stage, referred to as the chronic phase, which is clinically distinguished by a marked overproduction of granulocytes (Champlin and Golde, 1985). During this phase of the disease, Phl-positive cells become apparent in hematopoietic cells but not in other tissues (Rowley, 1975), and their numbers progressively increase. The chronic phase is unstable, and eventually patients experience either a shift to a progression of symptoms (the accelerated phase) or proceed directly to a more aggressive leukemia (the acute phase or blast crisis) characterized by the clonal malignant outgrowth of immature cells of either the myeloid or lymphoid lineage (Champlin and Golde, 1985). Progression to the blast crisis phase of CML is often accompanied by additional chromosomal abnormalities, including trisomy 8, isochromosome 17, or duplication of the Ph' chromosome (see Rowley, 1980). In the generation of the Ph' chromosome, the translocation breakpoint on chromosome 22 generally occurs within a limited 5.8-kb region, bcr, while the breakpoint on chromosome 9 occurs at a variable distance 5' to the c-abl gene (de Klein et al., 1982; Heisterkamp et al., 1983; Bartram et al., 1983; Groffen et al., 1984; Leibowitz et al., 1985b). The location of the c-abl gene close to the chromosomal breakpoint on chromosome 9 prompted the analysis of abl expression in Phl-positive CML cells. At the RNA level, it was noted that in addition to the normal c-abl transcripts, a novel large c-abl transcript was present both in Ph'-positive cell lines and clinical isolates (Canaani et al., 1984; Collins et al., 1984; Gale and Canaani, 1984; Leibowitz et al., 1985a; Stam et al., 1985).The Phl chromosome was also correlated with the presence of a novel c-abl-related protein (Konopka et al., 198413, 1985; Davis et al., 1985; Kloetzer et al., 1985; Konopka and Witte, 1985). It has also been demonstrated that a portion of Phlnegative CML cells actually contain complex chromosomal translocations which in turn generate both the CML-specific RNA and protein (Hagemeijer et al., 1984, 1985; Bartram et al., 1985; Teyssier et al., 1985; Morris et al., 1986; reviewed in Sandberg et al., 1986). Taken together, these results strongly implicate c-abl in the pathogenesis of CML. II. Structure and Expression of abl Genes

A. c-abl A c-abl gene has been detected in all vertebrate genomes tested (Goff et al., 1980). Molecular cloning and analysis of the human

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ANNE-MARIE MES-MASSON AND OWEN N. WITTE

(Shtivelman et al., 1985) and murine (Heisterkamp et al., 1983; Wang et al., 1984) c-abl genes indicate extensive similarities between these loci. In the human system, cDNA cloning of the c-abl mRNA reveals that the abl locus is composed of at least 11 different exons interspersed over at least 45 kb, which are brought together in the mature mRNA via RNA splicing (Shtivelman et al., 1985). Exons 1, 2, and a portion of exon 3 are unique to the c-abl gene and are not found in the v-abl protein (Shtivelman et al., 1985; Ben-Neriah et al., 1986a). In the murine system, cDNA clones indicate that four different forms of exon 1(types I-IV) can be spliced to the second or "common" exon of the c-abl gene (Ben-Neriah et al., 1986a). These results suggest the potential existence of four c-abl proteins, which differ from each other in their N-terminal sequences. What role these different forms have in determining subcellular localization and function remains to be determined. Currently, a single type of exon 1 has been described in the human system, and comparison of the nucleotide and amino acid sequences indicate that this exon is very similar to murine type I exon 1 (Shtivelman et al., 1985; Ben-Neriah et al., 1986a). The c-abl gene is expressed in most murine adult tissues but can be detected at a 3-fold higher level in spleen, thymus, and testes (Muller et al., 1982). Two c-abl mRNA species have been routinely detected in most cells: a 5- and 6-kb mRNA in murine cells and a 6- and 7-kb mRNA in human cells (Muller et al., 1982; Wang and Baltimore, 1983). There has been one unconfirmed report of four additional ablrelated transcripts in human hematopoietic cells (Savin et al., 1984). Although it was originally speculated that the various forms of the cabl RNA differed in their 3' untranslated sequences, recent evidence indicates that the difference is due to alternate exon 1 sequences at the 5' end of the mRNA. In particular, the 5-kb murine c-abl mRNA corresponds to RNA species bearing the type I exon 1, while the 6-kb mRNA probably represents type I1 and type IV exon 1(Ben-Neriah et al., 1986a). Despite this variety in c-abl mRNA species, only one electrophoretic species of the c-abl protein has been identified by imand a P145c-ablprotein in the munoprecipitation analysis, a P 150c-abl murine and human systems, respectively (Witte et al., 1979; Ponticelli et al., 1982; Konopka et al., 1984a). Both P145c-abland P150c-abldisplay in vitro tyrosine kinase activity, but a comparison of autophosphorylation sites indicates that the c-abl proteins differ from P160"-ablin the way they utilize themselves as substrates in the in vitro reaction (Konopka and Witte, 1985). Although P145c-ab2 and P150c-abz are phosphoproteins, neither is detectably phosphorylated on tyrosine residues in vivo (Ponticelli et aE., 1982; Konopka et al., 1984b). In addi-

abl

IN CHRONIC MYELOGENOUS LEUKEMIA

57

tion to the two c-abl mRNA species seen in most cells, a testes-specific 4.2-kb RNA has been found in stages of development after haploid reduction division, although its precise structure is not presently defined (Muller et al., 1982; Ponzetto and Wolgemuth, 1985).

B. P210c-ubl The Ph' chromosome results from the reciprocal translocation between chromosome 9 and 22. This translocation essentially fuses sequences at a variable distance 5' from the c-abl locus on chromosome 9 to sequences in a breakpoint cluster region (bcr) on chromosome 22 (de Klein et al., 1982; Heisterkamp et al., 1983; Bartram et al., 1983; Groffen et al., 1984; Leibowitz et al., 198513). It has been demonstrated that the bcr region falls within a gene coding region which is actively transcribed into a 4.5- and 6.5-kb mRNA species (Heisterkamp et al., 1985). The bcr gene is composed of at least 14 exons, and the Ph' translocation usually results in the loss of 6 or 7 of the 3' exons to chromosome 9 (Heisterkamp et al., 1985). The translocation of c-abl sequences downstream of bcr sequences leads to the altered transcription of the abl gene. Phl-positive cells revealed the presence of a novel large c-abl-related RNA transcript originally estimated to be 8.0-8.2 kb in size (Canaani et al., 1984; Collins et al., 1984). Partial cDNA cloning of the CML-specific RNA confirmed the hybrid nature of this RNA (Shtivelman et al., 1985; Grosveld et al., 1986). The 5' sequences of the RNA are derived from the bcr gene while 3' sequences represent exons 2-11 of the c-abl gene. There appears to be some microheterogeneity in the splice pattern at the junction of bcrabl sequences (Shtivelman et al., 1985). Full-length cDNA clones have recently been isolated and define the complete coding region of the bcr-abl sequences (Mes-Masson et al., 1986; see Section 111). An altered high molecular weight c-abl protein, P210c-QbL, has been detected in Phl-positive cells and clinical isolates (Konopka et al., 1984b, 1985; Davis et al., 1985; Kloetzer et al., 1985; Konopka and Witte, 1985). Experiments with mouse-human hybrids have indicated that the expression of P210c-"bldepends on the presence of the Phl chromosome and is not myeloid specific (Kozbor et al., 1986). P210c-"blcan also be expressed in B lineage lymphoid cell lines derived from Phl-positive CML patients, but at a lower and more variable level (Konopka et al., 1986). Comparison of P210c-"bzand the normal P145c-Qbl by two-dimensional peptide analysis indicates that the structural alteration in P210c-"blresults from the presence of nonabl polypeptide sequences N-terminal to c-abl-derived sequences

58

ANNE-MARIE MES-MASSON AND OWEN N. WITTE

(Konopka et aZ., 1984b). These N-terminal sequences are derived from bcr sequences as demonstrated by their ability to cross-react with bcrspecific antiserum (Ben-Neriah et al., 1986b). In contrast to the normal abl proteins, the P210c-ab2 protein is phosphorylated on tyrosine residues in vivo (Konopka et al., 1984b). In addition, P21WflbZ exhibits an in vitro tyrosine kinase activity remarkably similar to the P160v-abl activity, in that the altered c-abl proteins have similar optimal reaction conditions, thermal stability, and utilize similar tyrosine residues in the autophosphorylation reaction (Konopka et al., 1984b; Davis et al., 1985). A model detailing the events resulting from the generation of the Phl chromosome is presented in Fig. 1.Transcription initiates in chromosome 22, presumably from the bcr promoter although this has not been experimentally confirmed, and proceeds through the remaining bcr sequences, past the variable breakpoints in chromosome 9, and finally terminates in sequences downstream from the c-abl coding sequences. The intervening sequences, as well as the sequences 5' to the c-abl exon 2, are then removed via RNA splicing. The hybrid bcrprotein. abl mRNA is subsequently translated to yield the P210c--"b' Ill. Molecular Characterization of P210c-eb'

A. cDNA CLONING OF THE 8.5-kb mRNA To understand the role of P210c-ablin the progression of CML we felt it was necessary to obtain full-length cDNA clones of the entire

FIG.1. Schematic model for the production of the P210 protein from the Ph' chromosome. The approximate chromosomal structures for the c-bcr gene on chromosome 22 and the c-abl gene on chromosome 9 are shown. Exonic sequences are shown in the shaded or blackened boxes. Transcription initiates on chromosome 22 at an as yet unknown promoter and continues through the chromosomal breakpoint. The chromosomal breakpoint region is indicated by the double-headed arrow. The large primary transcript is spliced to a final RNA form of approximately 8.5-8.7 kb. This RNA encodes the P210, c-abl-derived protein. The 5' end of the large mRNA contains a G-C-rich sequence indicated by the unique slashed boxed area.

abl IN CHRONIC MYELOGENOUS LEUKEMIA

59

CML-specific mRNA. With these clones we would later be able to express P210c-Qbl in a variety of cellular backgrounds, allowing us to dissect the enzymatic activities and the physiological effects of this protein. Partial cDNA clones, extending from the 3' end of the mRNA past the bcr-abl junction, have previously been isolated (Shtivelman et al., 1985; Grosveld et al., 1986), and while these demonstrated the hybrid nature of the mRNA, they did not yield a clear picture of the final primary structure of P210c-ubl.We have succeeded in recovering overlapping cDNA clones which define the entire coding sequences protein; the construction and characterization of these for the P210c-Qb2 clones is discussed in detail elsewhere (Mes-Masson et al., 1986). However, a few of the details concerning the isolation and characterization of these clones is noteworthy. Since the bcr-abl mRNA is greater than 8 kb in size, we sought to recover large cDNA clones extending into the extreme 5' sequences. To this end, first strand synthesis was primed using both oligo(dT) and a specific internal oligonucleotide primer. The internal primer corresponded to the c-abl-conserved tyrosine kinase domain, thus positioning synthesis from the primer approximately 1 kb 3' to the bcr-abl junction. It was assumed that cDNA clones initiating from this oligonucleotide would only have to extend 4.5 kb in length in order to include all sequences at the 5' end of the bcr-abl mRNA. We also noticed that extensive denaturation of the RNA used to prime first strand synthesis resulted in the recovery of significantly larger cDNA inserts. In order to concentrate our efforts on large cDNA clones, double stranded cDNA was size selected over a sucrose gradient prior to ligation to A gtlO to enrich recovery of clones larger than 2 kb in size. A v-abl probe was used to initially screen cDNA libraries. Figure 2 indicates the recovery of v-abl hybridizing clones from cDNA libraries generated by using either poly(A+)K562 RNA or size selected (>4 kb) poly(A+)K562 RNA. The 10-fold enrichment of v-abl homologous clones allowed us to screen a large number (>300) of recombinant phage. Oligonucleotide probes corresponding to different regions of known bcr-abl sequences were used to map the location of a large number of cDNA clones (see Fig. 3). The most interesting clones were then extensively mapped and sequenced, and we presently estimate the bcr-abl mRNA to be 8.5-8.7 kb in size. The coding sequencing results provided us with information concerning both the 5' noncoding sequences (see Section II1,B) and the bcr-abl coding sequences (see Section 111,C). Using convenient restriction endonuclease sites, full-length bcr-abl cDNA clones containing the entire coding sequences for the P210c-abL have been constructed.

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FIG.2. Enhanced recovery of c-abl- and bcr-abl-derived cDNA clones from sizeselected RNA libraries. (A) Representative filter from the size-selected RNA-derived cDNA library. (B) Filter from a non-size-selected library. Each filter contained approximately 50,000 plaques. Both were screened with a nick-translated v-abl homologous probe representing the tyrosine kinase domain and central portion of the v-abl molecule. Approximately 10- to 20-fold enrichment for specific abl- and bcr-abl-related clones was achieved utilizing size-selected RNA.

B. 5‘ NONCODINGSEQUENCES Although we are at present uncertain of the exact extent of the 5‘ noncoding sequences in the bcr-abl mRNA, our sequencing results of the most 5’ cDNA clone reveals that these sequences extend over 400 bases in length. The 5’ noncoding sequences are greater than 80% GC rich. The role of the 5’ sequences is unknown, but, owing to the high G-C content, a number of stable secondary structures are predicted to occur in this region. In particular, using the programs of Zucker and Stiegler (1981), a 15-bp inverted repeat between nucleotides 78-92 and 343-357 provides the stem for a computer-generated stem-loop structure which has an estimated AG of -281 kcal/mol. We FIG.3. Screening stretegy for alignment of bcr- and abl-related cDNA clones. Phage miniprep DNAs were prepared and analyzed following restriction with endonuclease R1, which liberates the inserted fragment from the phage arms. Each set of DNAs was transferred by Southern technique to nitrocellulose filters and sequentially probed with end-labeled oligonucleotides representing sequences on the abl or bcr side of the chimeric mRNA junction. In this fashion, clones could be rapidly aligned for the relative sequence contribution from the abl and bcr gene, and extent of sequence homology approximated in a rapid fashion.

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have not determined if any secondary structures occur in vivo or in vitro, but it is interesting to note that a cluster of cDNA clones appear to have terminated 3’ to the location of a putative hairpin structure formed by the inverted repeats (Fig. 4).The influence of any 5’noncoding secondary structures on translation is uncertain, although it has been reported that the translation initiation complex has the ability to melt duplex structures in mRNA (Kozak, 1985).

C. CODING SEQUENCES We have identified an open reading frame in the 5’sequences of large bcr-ab2 cDNA clones initiating at the second AUG codon starting at nucleotide 471 and extending to nucleotide 2514 into the known bcr reading frame (Heisterkamp et al., 1985).Our sequence

GC INVERTED REPEAT

249 bp

+

GCCGCCGCCGCCGCC

c

PRIMARY STRUCTURE

......

K562cDNA I82

......

189

......

250

K562cDNA 16 1

I72 225

FIG.4. Hypothetical secondary structure of the bcr-abl mRNA generated by G-Crich inverted repeats near the 5’ end of the mRNA. The relative position of a perfect 15bp inverted repeat of all G-C sequence is shown. A representative sampling of cDNA clones which pass through this G-C-rich region, as well as a number which terminate at the base of the putative stem loop structure, are shown.

abl

IN CHRONIC MYELOGENOUS LEUKEMIA

63

results, in conjunction with previously published data (Heisterkamp et al., 1985; Shtivelman et al., 1985), reveals that the N-terminal 927 amino acids of the P210c-ab2 protein are derived from bcr sequences. The sequence of the subsequent c-abl-derived 87 amino acids has been previously identified (Shtivelman et al., 1985). Although the remaining c-abl sequences have not yet been reported, we can assume that they are similar to the published v-abl sequences (Lee et al., 1985), since antisera directed against the viral tyrosine kinase domain and the C-terminus of v-abl cross-react well with the c-abl proteins (Konopka et al., 1984a). From previously published bcr, bcrabl, and v-abl sequence (Mes-Masson et al., 1986; Heisterkamp et al., 1985; Shtivelman et al., 1985; Lee et al., 1985), a tentative nucleic acid and protein sequence for the P210'-""' protein can be assembled (Fig. 5). The first AUG identified in the bcr-ah1 mRNA is at position 366, and, although it is in the same reading frame as the second AUG, the first AUG is followed by an in-frame stop codon at nucleotide 420. The AUG at nucleotide 471 is the likely candidate to initiate translation, although we are uncertain whether the first AUG might be used in a different context, such as alternative splicing of the bcr-abl mRNA which might eliminate the stop codon at position 420 in the mature mRNA. We have as yet no evidence of alternate splicing, although ubl antisera also precipitate a P190 protein in Ph'-positive CML cells (Konopka et al., 1984b). The N-terminus of the bcr-ubl protein is predicted to be Met-Val-Asp and thus would not contain a Gly residue at position 2, a sequence associated with myristilation in a number of oncogenes including v-abl (Sefton et al., 1982). Using the proposed amino acid sequence of P210'-""1 (Fig. 5), a hydrophilicity plot can be deduced (Hopp and Woods, 1981; see Fig. 6). The C-terminus of the bcr-abl protein possesses the most hydrophobic profile, but it is unclear whether these sequences are sufficiently hydrophobic to direct stable association of the protein with membranes. It will be important to determine the precise subcellular localization of the bcr-abt protein, but preliminary results suggest that most of the protein is found in the cytoplasmic-soluble fraction (S. Lecker, S. Clarke, and 0. N. Witte, unpublished results). The amino acid composition of P210'-""' reveals a high (>lo%) serine content, but which of these residues is phosphorylated in vivo remains to be determined. IV. Future Directions

Cytogenetic studies in conjunction with glucose-&phosphate dehydrogenase isoenzyme analysis have demonstrated that CML is a clo-

50 CGGCTGGCTGAGCTTAGCGTCCGAGGAGGCGGCGGCGGCGGCGGCGGWG

I00

GCGACTACGIIGWCGCCWGTTWCCCCCGCTTCCTWGGAWCCTG

1250

lyAspTrrGluAspAlaG1uLruAsnProArgPhrLruLydspAsnLru CGGCGGCGGCGGGGCTGTGGGGCGGTGCGGWGCGAGAGGCGAGWGCGC

I50

GCGGGCCGTGGCtAtAGTCTGGCGGCGGCCTGGCGGAGCGGAGAGWGCG

200

CCCGCGCCTCGCCGTGCGWGGAGCCCCGWWCMTAGCGGCGCGCGW

250

GCCCGCGCCCnCCCCCCGGCGCGCCCCGCCCCGCGCGCCGAGCGCCCCG

300

ATCGACGCWTGGCGGTAGWGGCCCCCTTGGCCGCCCCTGWGTACCA

Il~AspA1.AsnG1rGlyScrArgProProTrpProProLruGluTyrGl GCCCTACWWGWTCTACGTCGGGGGWTWTGWGGGWGGGCMGG

1300

1350

nProTyrGlnScrI1rTyrV~1GlyG1~rtMrtGluG1rGluGlrLrsG I 400 1rProLruLru~rgScrGlnS~rlhrSrrGluGlnGluL~~r~L~uThr

GCCCGCTCC;GCGWGCW&GWCCTC~&GWGW&GCGC~AC~

350 GCCGCCGCGCGGGCCATGGGGGCCGCCCGGCGCCCGGGGCCGGGCCTGGC

400

GAGGCCGCCGCGCCGCCGCTUIGACGGGCCCCGCGCGCAGCCCGGCGGCG

4SO

tAGGTPAGGCCGGCCGCGCtTGGTGGACCCGGTGGGlXTCGCGWGGCG nrtU.lAspProuilGlrPhrn1 r G l u A l a

500

TGGMGGCGCAGTTCCCGWCTWWGCCCCCGCGCATGWGCTGCGCTC

550

AGTGGGCWWTCWGCGWGCfGGAGCGCfGCMGGCCTCWTTCGGC

600

TrpLrrCIlrGlnPhrProAspSrrGluProProAr#lrtGluLruArgSr

TGGCCCCGtAGGTCCTACTCCCCCCGWGTTTWGWTTGCGWGGCGG

1450

ACTKTCCTCTGGCCAGTCCGCCGCGTGTCCCCMGCCCWCWCCTAC

1350

TrpProArp9rgSrrTrrSerProArgSrrPh~GluAspCrsGIrGlrGl

spPhrSrrSirGIyGlnScrSrrArgV~1SerProSrrProThrlhrTyr

rV~1G1rA~pflrG1uG1nGluLruG1uArpCy~Lr+CII~SrrIlrAr~

GCCTGWGCAGWGQTW~WGWGCGC;TCCGWTW~CTACCTGW~ 650 ACTGCCCGGTTGTCGTGTCCGAGGCCACGTCGTGGGCG~ I 700 rgLruG1u0lnGluV~1A~nG1nG1uArgPhrAr#l~tllrTyrLruG1n i~CrrPr~~1Vr1V~1SrrGluAlrThrllrU~lGlrV~1ArpLy~Thr

ACG~TGCTGGCC~~GGWWGC~ATWCCGGWGCW~GGGG~ ThrLeuLeuAl rLysG1 uLrrLr,SrrTyrA,pArgGl nArgTrpGl rPh

700

GG6CAWTCIGGCCCMCCLYTGGCWGGGCGCCnCWTGWCQCGCAGA

1750

GlyGlnllrTrpProA~rw4~pGlrGluGlyAlrPh~H~rGl~~pAI~~

CCGGCGCGCGGCGCAGGCCCCCGACGGCGCCTCCGAGCCCCGAGCGTCCG 750 rAr~rpCIlrAlrGlnAl~Prc4spGlrAlrS~rGluPrc4rpCI1~SrrA

CGTCGCGCCCGCAGCCAGCGCCCGCCGACGGAGCCGACCCGCCGCCCGCC

800

GAGGAGCCCGAGGCCCGGCCCGACGGCWGGGllCTCCGGOTfWGGCCAQ GI uGluProGl uAl drgPrc4spGl rGluGlrSirProGlrLrlA1 d r

850

GCCCGG WCCGCCCGCAGGCCCGGGGCAGCCGCGTCGGGGWCGGWCQ gProGl rlhrnl aArg4rgProGl HI rAl .A1. S i r G I yGluArg4spA

900

lrSrrArgProGlnPra4lrPra4I.AspGlrAl.AspProProProAlr

TGGCCGTTCGGMCACCACCTG@+TACGGCTGCGCTGCAGACCGGGCAG

1800

pGlrSirPhcG1rThrProProGlrTrrG1yCr~l~I1ArpArg4lrG 1850

TCCTCATCGCCCMCCTCAGCAGUWGGGCAGGGGCAGCCGGGATGCGCT

1900

ScrS~rSrrProHisLruSrrSrrLyrGly~rgGl~SrrAr~spAlrLr

1 vso

ACCGGGGACCCCCCGCCAGCGTGGCGGCGCTCAGGTCWCTTCGAGCGQ 950 spArgGlrProPr~lrSirV~1Al.CI1~LiuAr~SrrAsnPh~G1uAr~

GGGCTTGGAOAGTW0TGGGTCCTGTCGGWTCCTGGCTAGCGAG 2000 r~lrTrpArgV~1ArgLrsTrpU.1LiuSirGlrllrLruAlrSirGlu

1000

2050

1050

21 00

iiao

2130

1150

2200

FIG.5. Compiled cDNA sequence of the known bcr-abl sequence. Sequence results from our present study, in concert with previously described sequence from the work of others (see text for references), and the known sequence of the v-abl oncogene were used to compile a putative sequence for the entire bcr-abl gene. The anticipated differences between the human c-abl and murine v-abl sequences are appreciated, but at this time a complete human c-abl sequence is not available. The putative open reading frame initiates at position 471 and extends through nucleotide 6353. Untranslated 3' sequences of the murine gene are not shown. (Continued on pp. 66 and 67.)

GGGCWCCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCT 1 GlrAspLruPhrGlnLvsLruAl r S r r G l n L r u G I r V . l T y r A r ~ 1 rP

2250

k 4 k a B C C C C A Q C G O C C A O G C A T C T W ~ W G C C T C A G G ~ C T W 3300

2300

GTGMGCCGCTCF1TGtCTCWGMCCCTCGCTGWCCCAW rrQlUAl d l .ClrpTrpAsnSrrLrsGl uAsnLruLruAl a01 rProSrr

2350

rLy~l~LruGlnArpProV~lAl~SrrAspPhrQ1uProG1nG1rL~u~ 3350

~ T G A C C C t C I A C C C F 1 T G C A C T G T A T W ~ G T G G C C A ~ G Q3400 G1uAsnAspPr~snLruPh~U~lAl~LruT~rAspPhrU~1Al~S~rGl

3450 2400

3500 24SO

3550 2500 o? o?

CTGGTATCATGGCCCTGTATCTCGGMTGCTGCTWGTATCTGCTGAGCA

3600

CCTCCGCATCTCACAGAACCCTGTCCAGCATtCIATWGWWTCACAC

zsso r T r p T y r H i ~ G l y P r o V ~ l S r r A r g 4 s n A I ~ l ~ G l u T y r L r u L ~ u S r r S

CCCWCGGCAGTCCATGACGGTGMGMGGWWGCACCGGCAGCTGCTG

2600

~LruArgllrSrrGlnArnPhrLruSrrSrrIlrAsnGluGluIlrThrP

3650

r~r~rpGlnSrr~rtThrU~lLysLrsGlrGluHiIC\rpGlnLruLru

3700 AAGWCAGCCATGGTGGAGCTGGTGWGGGGGCCCGCMGCTGCGCCA

LrsAspSrrPhrHrtU~lGluLruU~lG1uG1rC11~rgLysLruArgHi

2650 3750

C G T C T T C C T O r r C A C C W C C T G ~ C T C T G C A C W G C T t C I A G M G ~ W 2700

sU.1PhrLruPhrThrAspLruLruLruCysThrLysLruLysLysGlnS GCGWGGMCGCAGCAGTATWCTGCCUYITGGTACAlTCCGCTCACQ rrGlrGlvLrsThrGlnGlnTyrAspCv,Lr,TrpTyrIlrProLruThr

3800 2750

. . .

GATCTCAGCTTCCAWTGGTGGATGMCTGGAGGCAGTGCCWCATCCC

AspLruStrPhrGlnMttU~lAspGluLtuGluAIrU~lProlisnIltPr

2800

AtCACCACACTCCACTACCCAOCTCCWGCGWWGCCCA~ATCTA IlrThrThrLruHisTyrPr~Al.ProLrdr~gnLrrProlhrl1 t T r

3850

CGBTOTGTCCCCCMCTACGAWGTGGWTGMGCGCACCGACATm

3900

2850

rGlrV~lStrPraAsnTrrAspLysTrpGl~rtQl~rgThrAsp11~T

2900

CCATGMGCAWS'TTGBQTGGAGQCCAGTACGGGGAGQTGTACGAGGGC

3950

hr~rtLysHisLysL~uQlyQlyQlyQlnTyrGlyGluU~lTyrGluGly 2950

F m G O I Y I ~ O T A C A G C C T C A C T G I G G C C G T ~ G A C ~ G M G G A G4000 ~

U~lTrpLy~LrsTyrS8rLruThrU~lAl~U~lL~sThrLruL~5GIuAs

3000

CACCATGGAGBTGWGGAGlTCCTGMGWGCGGCGGTGATGMGGAGA

3050

T C A C I A C C I C C C T A C I C C T G O T G C A G ~ G ~ A G G G O T G T G 4100

31 00

C C A ~ C T A W T A C I T C A C T G A G n C A T C C I C C T C I T O O U Y l t A 4150

31SO

C C T ~ G G G A G T G T A A C C Q G C A G G A G ~ G A G C G C C ~ G ~ ~ C T G ~4200 CTA~ rLruArgGluC~drnArqQlnGluU~1StrAl ~U~lUalLtuLtuTrrn

3200

T G G C C A C A C A ~ T C T C A T C A G C C A T G G A G T A C T T G G A G M ~ ~ C T T C4250

32SO

A T C C A C A G A O A C C I T G C T G C C C G W C T G C C T G G T A G G G ~ C C A ~ 4300

4050

pThrMrtGluU~1GluGluPhrLtuL~sGluAldl~V~l~rtL~sGluI

1rL~sHisProli~nLruU~1G1nLruLruGl~U~lC~sThrArqGluPro

ProPhtT~r11t1ltThrGluPh~rtThrTyrG1rClsnLruL~uAspT~

rtAl~ThrGlnIltSrrStrA1aIlrtGluTyrLtuGluL~sL~dsnPhr

I1rHidr~spLruAI~lrClr~snCysLtuU~lGlyGluA~nHi~Lr

FIG.5. (Continued)

G G T O n n G G T G G ~ U l ~ G G C t T t n G M G G R G A T G A M G G G G A M C C T 4350 uUAlLvsUAlAl.ArpPhcG1rL~uScrArgL~uMct~hrGl~spThrf

ACXGGCCMTGCTGIXGCCCCCMTWTGGACCGMCCTWG

4400

YrThrAlAHidlAGIrdlALysPhiProllrLysTrpThrAl~ProGlu

AGCCTGGCCTAMMGTrCTCMTMGTCGGACFTGTGGGCGG

~crLcuAlATrrAsnLyrPhrSerlliLysSerAspUAlTrpAlAPhiG1

8 W t C A ~ A G T G G C G G T C G F T M C G ~ G C C T C ~ ~ C C T G C C F T ~ G C5400 TG g4spThrGluTrpArgSrrU~lThrLiuPr~rg4spLiuProSirAlAG

G M G C A O m t C \ C T M T C C C ~ G G A G G G M ~ G C W G C M5450 lyLysGlnPhiCIspSirSirThrPhiGl~Gl~HisL~sSirGluL~sPro

4450

GCTCTGCCTCGGk4ACGMCOIAGT~OCIGMG~CTGAGMGAGTGGCM 5500 Al~LiuPrdlrgL~drgThrSirQluSirArgSrrGluGlnU~l~l~L~ 5550

5600

5650

5700

5750

5800

5850

5900

4900

5950

4950

6000

6050 5000

Q,

W

6100

5050

TCGnCGGCTMGCtTCAGGGCIIXCCCACCAGCCCGGTCTCCACCCCCTCC SrrThrAl &LysProGlnGlYLruProPrmll .ArpSrrProProProPr

5100

CICAGCACCAGCTCCTTCACCCCTGGCTGGGWCCAGCAGCCATCTTCTGC

6150

ATGGGCICCACCAGCTCTCACCTCAGACGCAGCAGCIGCCTACWGTCCCC 51 50 snGlyThrThrSrrSrrHisLruCIr~rgScrCIr~l~TyrGlnUalPro

CGCCTTCATCCCCCTCATATWCCCUGTGTCTCTCIGWGCICCCGCC

6200

aPnGGCCAGWTGGGGCTGGCTCCtTMTGGAGCCTTCCGGGCIGCCGGG

5200

AGCCGCCAGCIGCGCATTGCCAGTGGCACCATCACCMGGGTGTGGTTCTG

6250

WCTCAGGCTTCCGTTCTCCCCACATGTG~GTCCAGCACAIXGCI

5250

GACAGTCICTGAGGCC~TGTGCCT~GCCAT~TCCCGWC-~CAGAGCAGA~

6300

LrsGlyGlnGlnTrpGlrTrpLruProCIsnGlyCII~PhrArgGluProGl

yCIsnSrrGlrPhcArgSrrProHi31rtTrpLrrLrsSrrSrrThrLruT

CCGGGCIGCCGCCTGGCTG~GCCWAGAG&K~AGCGGCA~AGCTCCA&

hrGlrS~rArgLruCIlrCIlrCIlrGluG1uG1uSrrGlrClrtSrrSrrSrr

. .

oGlnHisGlnLcuLruHirProTrpLcuGlrThrSrrSrrHisLruLruP

roProScrSrrProScrTrrGlnPrdJ~1CrrLruLruGlrCIrgProCIl~

~rrclrgGlnScrCI1rLruPrdJIlAl~ProScrProCIrpV~~~rpPhrfr

pThrUa1LruCIrgProCy~AI.LluProSrrProGlrThrGlnSrrArgT

5300

MGCGClTCCTGCGff CTr GTTCGGCCTCCTGCATGCCCCATGGGGWG 5350 Ly~rgPhrLruArgSrrCyrSrrlll~SrrCr31rtProHirGl*Al~r

G G C C A G C C A ~ ~ G T G C S G T A C T G O A G O C T G ; ~ W W C ~ ~ C I C6350 A~~ rpPr~lrThrUr1L~uTyr~rpCIrg~~uCIlrCIrgThrCrrlhrLruScr

GTGTM

Val End

FIG.5 . (Continued)

6356

70

ANNE-MARIE MES-MASSON AND OWEN N. WITTE

--

+3-

-

hydrophi I i c ........................................................... I

I

-

-3

: - ........................................................... --

hydrophobic

fraction o f length

FIG.6. Hydrophilicity plot of the putative bcr-abl sequence. Using the Hopp and Woods program we have constructed a hydrophilicity plot of the putative bcr-abl sequence. The protein is generally hydrophilic and shows only short stretches of contiguous hydrophobic characteristic.

nal disorder involving the hematopoietic stem cell (Fialkow et al., 1981; Martin et al., 1982). Since a clinical picture of CML can be manifested before the appearance of the Ph' chromosome (Hayata et al., 1975; Lisker et al., 1980), it has been suggested that chromosomal translocation is not the primary event that affects the stem cell in CML. While the bcr-abl-specific mRNA and protein probably play a secondary or complementary role in the progression of CML, it is probably not sufficient to cause the fulminating disease. In particular, a number of additional abnormalities have been observed in the blast crisis phase of the disease. These include additional chromosomal rearrangements, such as trisomies, duplication of the Ph' chromosome, and complex rearrangements (Spiers and Baikie, 1968; Rowley, 1975; see Champlin and Golde, 1986). In addition it has been reported that expression of c-sis, located on the translocated arm of chromosome 22 not present in the Ph' chromosome, increases during blast crisis (Romero et al., 1986), although the significance of this elevated expression is unknown. These results seem to suggest that the generation of the Ph' chromosome is a necessary but not sufficient step in the full progression of CML. Whether the bcr-abl sequences are required to establish conditions leading to blast crisis or whether expression of the bcr-abl protein needs to be maintained during blast

abl

IN CHRONIC MYELOGENOUS LEUKEMIA

71

crisis remains to be determined. The precise role of the abl gene in normal and neoplastic transformation remains to be determined. One important approach involves the continued characterization of all forms of abl protein, including identification of the substrates and the factors that modulate abl kinase activity. A second approach, made possible by the cloning of the coding sequences for P210c-abz, are gene transfer experiments which will allow the expression of the bcr-abl protein in a variety of cellular backgrounds. This type of analysis should provide some indication of the role of P210"-ab'in CML.

NOTEADDEDIN PROOF Since the manuscript was prepared, the primary structure of two forms of the human c-abl protein, differing only in the use of alternate exon 1 sequences, has been described (Shtivelman et al., 1986). Although this new information alters some of the compiled bcr-abl cDNA sequence (Fig. 5), it does not significantly affect the overall primary protein structure or the hydrophilicity plot (Fig. 6) of the bcr-abl protein.

ACKNOWLEDGMENTS We gratefully acknowledge Carol Crookshank for assistance in preparation of the manuscript and Kathleen Denis for assistance with DNA sequence handling. A.-M.M-M. is supported by a Research Fellowship from the National Cancer Institute of Canada. O.N.W. is a Faculty Scholar of the American Cancer Society. Work reported from our laboratory was supported by grants from the National Cancer Institute to O.N.W.

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ubl

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Martin, P. J., Najfeld, V., and Fialkow, P. J. (1982). Cancer Genet. Cytogenet. 6, 359368. Mes-Masson, A.-M., McLaughlin, J., Daley, G. Q., Paskind, M., and Witte, 0. N. (1986). Proc. Natl. Acad. Sci. U.S.A. 83,9768-9772. Morris, C. M., Reeve, A. E. Fitzgerald, P. H., Hollings, P. E., Beard, M. E. J., and Heaton, D. C. (1986). Nature (London) 320,281-283. Muller, R., Slamon, D. J., Tremblay, J. M., Cline, M. J., and Verma, I. M. (1982).Nature (London) 299,640-644. Nowell, P. C., and Hungerford, D. A. (1960).J. Natl. Cancer Inst. 25, 85-109. Ponticelli, A. S., Whitlock, C. A,, Rosenberg, N., and Witte, 0. N. (1982). Cell 29,953960. Ponzetto, C., and Wolgemuth, D. J. (1985). Mol. Cell. Biol. 5, 1791-1794. Prakash, O., and Yunis, J. J. (1984). Cancer Genet. Cytogenet. 11,361-367. Prywes, R., Foulkes, J. G., Rosenberg, N., and Baltimore, D. (1983). Cell 34, 569-579. Prywes, R. J., Foulkes, J. G., and Baltimore, D. (1985).J. Virol. 54, 114-122. Reynolds, F. H., Sacks, T. L. S., Deobagkar, D. N., and Stephenson, J. P. (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 3974-3978. Reynolds, R. K., Van de Ven, W. J. M., and Stephenson, J. R. (1980).J. Virol. 36, 374386. Romero, P., Blick, M., Talpaz, M., Murphy, E., Hester, J., and Gutterman, J. (1986). Blood 67,839-841. Rosenberg, N., Baltimore, D., and Scher, C. D. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 1932-1936. Rosenberg, N., Clark, D. R., and Witte, 0. N. (1980).J. Virol. 36, 766-774. Rowley, J. D. (1975).Proc. Natl. Acad. Sci. U.S.A.72, 152-157. RowIey, J. D. (1980). Annu. Rev. Genet. 14, 17-40. Sandberg, A. A., Gemmill, R. M., Hecht, B. K., and Hecht, F. (1986). Cancer Genet. Cytogenet. 21, 129-146. Savin, K. W., Adams, F. C., Devercux, L. M., Jose, D. G., and de Kretser, T. A. (1984). Mol. Biol. Med. 2, 397-409. Scher, C. D., and Siegler, R. (1975). Nature (London) 253,729-731. Sefton, B. M., Hunter, T., and Raschke, W. C. (1981). Proc. Natl. Acad. Sci. U.S.A.78, 1552-1556. Sefton, B. M., Trowbridge, I. S., Cooper, J. A., and Scolnick, E. M. (1982). Cell 31,465474. Sefton, B. M., Hunter, T., and Cooper, J. A. (1983). Mol. Cell. Biol. 3,56-63. Shields, A., Goff, S. P., Paskind, M., Otto, G., and Baltimore, D. (1979). Cell 18, 955962. Shtivelman, E., Lifshitz, B., Gale, R. P., and Canaani, E. (1985). Nature (London) 315, 550-554. Shtivelman, E., Lifshitz, B.,Gale, R. P., Roe, B. A,, and Canaani, E. (1986). Cell 47,277284. Spiers, A. S. D. (1977). Clin. Haematol. 6, 77-86. Spiers, A. S. D., and Baikie, A. G. (1968). Br. J . Cancer 22, 192-204. Srinivasan, A., Dunn, C. Y., Yuasa, Y., Devare, S. G., Premkumar, E., Reddy, E. P., and Aaronson, S. A. (1982). Proc. Natl. Acad. Sci. U.S.A. 79, 5508-5512. Stam, K., Heisterkamp, N., Grosveld, G., de Klein, A., Verma, R. S., Coleman, M., Dosik, H., and Groffen, J. (1985). N . Eng1.J. Med. 313, 1429-1433. Teyssier, J. R., Bartram, C. R., Deville, J., Potion, G., and Pigeon, F. (1985). N . Eng1.J. Med. 312, 1393-1394.

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Van d e Ven, W. J. M., Reynolds, F. H., Jr., Nalewaik, R. P., and Stephenson, J. R. (1979). J. Virol. 32, 1041-1045. Van de Ven, W. J. M., Reynolds, F. H., Jr., and Stephenson, J. R. (1980). Virology 101, 185-197. Wang, J. T. J., and Baltimore, D. (1983). Mol. Cell. Biol. 3, 773-779. Wang, J. Y. J., Queen, C., and Baltimore, D. (1982).J. Biol. Chem. 257, 13181-13184. Wang, J. Y. Y., Ledley, F., Goff, S., Lee, R., Groner, Y ., and Baltimore, D. (1984).Cell 36, 349-356. Watanabe, S. M., and Witte, 0. N. (1983).J. Virol. 45, 1028-1036. Witte, 0. N., Rosenberg, N., Paskind, M., Shields, A., and Baltimore, D. (1978). Proc. Natl. Acad. Sci. U.S.A.75,2488-2492. Witte, 0. N., Rosenberg, N., and Baltimore, D. (1979). Nature (London) 281,396-398. Witte, 0.N., Goff, S . P., Rosenberg, N., and Baltimore, D. (1980a).Proc. Natl. Acad. Sci. U.S.A. 77,4993-4997. Witte, 0. N., Dasgupta, A., and Baltimore, D. (1980b). Nature (London) 283, 826-831. Zucker, M., and Stiegler, P. (1981). Nucleic Acids Res. 9, 133-148.

THE EPSTEIN-BARR VIRUS AND THE IMMUNE SYSTEM Giovanna Tosato Division of Biochemistry and Biophysics, Center for Drugs and Biologics, Food and Drug Administration, National Institutes of Health. Bethesda, Maryland 20892

1. Introduction

One of the major reasons for interest in the Epstein-Barr virus (EBV) is its strong association with two forms of human cancer, African Burkitt’s lymphoma and nasopharyngeal carcinoma. Although a number of viruses are known to cause tumors in animals, only two such agents, EBV and the human T cell leukemia virus type 1 (HTLVI), have a link to human cancer (for review, see Klein and Klein, 1985). EBV was first described in 1964 by Epstein et al. as a herpesvirus particle visualized on electron microscopic examination of cultured Burkitt’s lymphoma cells (Epstein et al., 1964). Since that time major advances have been made in the understanding of the biology of EBV, and a whole spectrum of disorders have been linked to this virus. It is now recognized that this herpesvirus is ubiquitous and that it asymptomatically infects most adult individuals worldwide (Henle and Henle, 1979a). Evidence of EBV infection is found in peripheral blood, where a small proportion of the B cells are latently infected with the virus (Diehl et al., 1968; Gerber et al., 1969; Nilsson, 1979; Tosato et al., 1984), and in the oral cavity, where the virus is intermittently shed (Gerber et al., 1972; Miller et al., 1973; Chang et al., 1973; Yao et al., 1985). Epithelial cells of the oropharynx, ductal cells of the parotid gland, and the lungs have been reported to be sites of EBV replication (Morgan et al., 1979; Sixbey et al., 1984; Lung et al., 1985), and the virus present in buccal fluid is believed to be responsible for horizontal transmission of infection. Primary infection with EBV is generally asymptomatic, particularly in childhood, but has a 50% chance of causing acute infectious mononucleosis in young adult and adult individuals (Evans et al., 1968; Henle et al., 1968; Niederman et al., 1968; Sawyer et al., 1971; Henle and Henle, 1979a). Occasionally, EBV-induced acute infectious mononucleosis has been followed by the appearance of both hypo75 ADVANCES IN CANCER RESEARCH, VOL 49

Copyright Q 1987 by Academic Press, Inc All rights of reproduction in any form reserved

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gammaglobulinemia and malignant polyclongal lymphoproliferation (Provisor et al., 1975, Purtilo, 1980; Greally et al., 1983). The latter syndrome has recently been the object of much interest, since it has occurred almost entirely in individuals with an underlying immunodeficiency. Some of these patients are male with a familial X-linked disorder, the nature of which is still to be defined (Purtilo et al., 1975, 1982; Sullivan et al., 1983).Other cases occur sporadically in children or young adults with presumed congenital defects in cellular immunity (Britton et al., 1978; Robinson et al., 1980; Fleisher et al., 1982). Finally, a number of cases have been observed in individuals receiving immunosuppressive regimens with agents such as cyclosporin A and anti-T cell receptor monoclonal antibody (Borzy et al., 1979; Calne et al., 1979; Crawford et al., 1980; Thiru et al., 1981; Hanto et al., 1982; Martin et al., 1984). In addition to these illnesses, recent attention has been given to a group of patients who, following primary infection with EBV, developed a chronic and disabling atypical illness associated with a characteristic serum antibody reactivity to EBV (elevated antibody titers to EBV early antigens and/or absent reactivity to EBNA, the EBV-related nuclear antigen) (Tobi et al., 1982; Straus et al., 1985; Jones et al., 1985). This newly described and still incompletely defined syndrome has been named chronic active EBV infection, or chronic symptomatic EBV infection, or chronic infectious mononucleosis (Tosat0 et al., 198513). The original discovery of EBV in African Burkitt’s cells was not accidental, and much evidence has been accumulated demonstrating a striking association of this virus with this rare neoplasm (Burkitt, 1963). For example, the vast majority (98%) of African Burkitt’s lymphomas are infected with EBV, and in the endemic areas elevated serum antibody titers to EBV-specific antigens predict the occurrence of the lymphoma (zur Hausen et al., 1970; de The et al., 1978). However, tumors that are indistinguishable from the African type occur sporadically, and only about 20% of these cases contain EBV (Anderson et al., 1976; Olweny et al., 1977; I.T. Magrath, 1982). In addition, B lymphocytes infected in vitro with EBV may become immortalized, but they lack certain features of Burkitt’s cells, including the characteristic chromosdmal translocations involving the cellular myc oncogene in chromosome 8 (Bernheim et al., 1981; Dalla-Favera et al., 1982, 1983; Nilsson and Klein, 1982; Klein, 1983). These facts have led many investigators to believe that the occurrence of endemic Burkitt’s lymphoma may require multiple interdependent steps, an essential one being B cell infection with EBV (for a review, see Klein,

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1981). New interest in the role of EBV in Burkitt’s lymphoma has recently arisen because an increasing number of patients with the HIV (human immunodeficiency virus)-induced acquired immunodeficiency syndrome have developed EBV-positive Burkitt’s lymphomas (Ziegler et al., 1982, 1984). It is hoped that in the context of this syndrome new elements will emerge that will help to clarify how Burkitt’s lymphoma is generated. Regarding the other tumor linked to EBV, nasopharyngeal carcinoma, the virus is implicated with the undifferentiated form. This association is true for tumors from all parts of the world since 100% of these ubiquitous carcinomas carry EBV DNA in the malignant epithelial cells (Old et al., 1966; Henle et al., 1970; Desgrandes et al., 1975; Anderson-Anvret et al., 1977; Huang et al., 1978). In addition, it is well established that in southern China and southeast Asia, where nasopharyngeal carcinoma is at high risk, serum IgA antibody titers to the capsid antigen of EBV (IgA-VCA)are much higher than in control groups. Also, elevated IgA antibody titers to VCA closely correlate to pathological changes in the nasopharynx (Zeng, 1985). Because of these remarkable findings, it is believed that EBV is likely to play a unique causative role in this tumor (Epstein, 1984). Thus, EBV that originally was detected in association with a rare cancer of African children turns out to be a ubiquitous and mostly innocuous agent for most humans. Although relatively rare, however, a whole spectrum of disorders have been causally linked to EBV, including acute infectious mononucleosis, hypogammaglobulinemia, malignant polyclonal B cell proliferation, chronic active-symptomatic EBV infection, African Burkitt’s lymphoma, and nasopharyngea1 carcinoma. This variety of possible outcomes associated with EBV infection is at first somewhat puzzling and confusing. With the exception of nasopharyngeal carcinoma, however, all other conditions can be related, perhaps simplistically, to the property of EBV to induce B cell immortalization and to the capacity of the immune system to recognize EBV-infected B cells. In this article, I shall summarize studies focused both on the effects of EBV on human B cells and on the immune response to viral infection, and I shall discuss how this information has helped us to understand the diverse aspects of virus-host interaction. II. The Virus

Similar to other herpesviruses, the complete particle of EBV is composed of a core consisting of Iinear double-stranded DNA, an icosahe-

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dral capsid, and a membrane envelope that encloses the capsid. During the last few years the entire EBV genome has been cloned (Dambaugh et al., 1980; Raab-Traub et al., 1980), and the DNA sequence of a prototype strain, B95-8, has been reported (Baer et al., 1984). The genome of EBV is approximately 172 kb (kilobases) long and consists of unique and tandemly repeated DNA elements. The general organization of EBV DNA is shown in Fig. 1.There are multiple terminal repeat (TR) sequences at each end of the DNA approximately 500 bp (base pairs) long; the number of these repeats varies among different EBV isolates and among molecules from each isolate (Given et al., 1979; Kintner and Sugden, 1979). Although the functions of the terminal repeat sequences are not fully understood, one reported role is to permit circularization of EBV DNA. It is known that the viral genome is linear in the virus particle but exists as a circular episome inside the infected cell (Pritchett et al., 1975; Hayward and Kieff, 1976; Lindahl et al., 1976). Here the terminal repeat sequences at opposite ends of the DNA become covalently linked, and it is believed that the repeats may facilitate circularization by enabling base pairing (Dambaugh et al., 1980). Four direct tandem internal repeat sequences (IR) have been identified; IR1 consists of multiple repeats of a 3072-bp sequence, IR2 and IR4 are tandem repeats of sequences of 125 and 103 bp, respectively (Cheung and Kieff, 1982; Dambaugh and Kieff, 1982). IR3 represents a sequence of 708 bp that consist of a repeat array of three nucleotide

Linear

EBV DNA u2

IR3 I

U4

I R 4 U5 TR I

I

Id 3

FIG.1. Epstein-Barr virus DNA. Schematic representation of linear EBV DNA and of circular EBV DNA episomes showing the general organization of unique (U) and major tandem internal repeat (IR) DNA domains.

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triplets, GGG, GCA, and GGA (Heller et al., 1982). The internal repeat sequences separate the EBV genome into 5 unique regions (U); U1, U2, U3, U4, and U5 are largely unique DNA domains of 10,3,59, 40, and 30 kb, respectively. The U2 region varies extensively among different EBV isolates and is almost entirely deleted in the P3HR-1 strain of EBV (Raab-Traub et a,?.,1978; Bornkamm et al., 1982; King et al., 1982; Dambaugh et al., 1984; Jeang and Hayward, 1984). Analysis of restriction endonuclease fragments of different EBV isolates obtained from various geographic and pathological origins has revealed the existence of polymorphism among EBV DNAs (Bornkamm et al., 1980). This polymorphism is sufficient to identify each isolate, but no feature has emerged to permit a correlation between a certain restriction pattern and a geographic source of the virus or type of disease (Raab-Traub et al., 1980; Heller et al., 1981; Bornkamm et al., 1984; Pollack et al., 1984). Therefore, these studies do not permit one to associate a particular EBV subtype with a single disease manifestation. However, since the level of resolution of comparative restriction endonuclease studies is limited to several hundred nucleotide pairs, only future investigation will permit one to assess the issue of EBV subtypes adequately (Kieff et al., 1983). Most EBV-infected B cells harbor the virus in a latent form. The frequency of spontaneous permissive infection varies in different human B cell lines but is believed to range only between 0 and 3%; marmoset B cell line are somewhat more permissive, and up to 10%of the cells may replicate the virus (Miller and Lipman, 1973a,b; Sugden et al., 1979). It is known that in most instances latently infected cells contain multiple copies of the EBV genome and that the entire EBV DNA is usually present (Nonoyama and Pagano, 1971; zur Hausen et al., 1972; Kawai et at., 1973; Pritchett et al., 1976; Sugden et al., 1979; Matsuo et al., 1984). In nonpermissively infected B cells, the EBV genome is likely to be replicated by a cellular polymerase since treatment of EBV-infected cells with agents that functionally inactivate the viral DNA polymerase, such as phosphonoacetic acid and acyclovir, does not affect latent EBV DNA replication (Summers and Klein, 1976; Colby et al., 1980; Sixbey and Pagano, 1985). Studies of the physical state of EBV DNA in nonpermissively infected cells has revealed that most of the genome is not covalently linked to cellular DNA but is maintained as circular episomes (Nonoyama and Pagano, 1972; Lindahl et al., 1974). A number of studies have examined whether or not EBV DNA is integrated at all into cell DNA (Nonoyama and Pagano, 1972; Adams et al., 1973). Recently, analysis of Namalwa and IB4 cells has demonstrated unequivocally

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the presence of integrated viral DNA in these cell lines (Kieff et al., 1984; Matsuo et al., 1984). In particular, while IB4 cells harbor EBV DNA in both episomal and integrated form, Namalwa cells contain only integrated EBV DNA, suggesting that integration is the only mechanism of viral persistence in these cells. It is interesting to note that EBV DNA is integrated into chromosome 4 of IB4 cells but into chromosome 1of Namalwa cells, suggesting that while viral DNA has a preferred site of integration within certain cells, this may differ from cell line to cell line (Henderson et al., 1983). This variability of integration site has increased difficulty in the understanding of the biological significance of EBV integration into cellular DNA. B cells nonproductively infected with EBV express a virally encoded nuclear antigen, EBNA, recognized by indirect immunofluorescence (Reedman and Klein, 1973). In addition, it is generally believed that B cells latently infected with the virus express a cell surface antigen (or antigens) named LYDMA (lymphocyte-determined membrane antigen) recognized by specific cytotoxic T cells (Svedmyr and Jondal, 1975). Recent studies of B cells latently infected with EBV have revealed that the virus is responsible for the expression of at least four distinct proteins, three of which are nuclear and have been named EBNA 1,2, and 3 and one a membrane protein named LIMP (latent infection membrane protein). The messenger RNA encoding three of these proteins, EBNA 1, EBNA 2, and LIMP, is transcribed from separate regions of the viral genome that have been named LTl , LT2, and LT3 (Kieff et al., 1984). This was conclusively demonstrated by raising antisera and monoclonal antibodies to the recombinant proteins encoded by portions of each of these genes that specifically reacted to distinct antigens in cells latently infected with the virus. The region of the EBV genome that may encode for the third nuclear protein, EBNA 3, has been tentatively identified and named LT4. The LTl gene consists of EBV DNA identified as U2 and the adjacent IR1 domain (Dambaugh et al., 1979; Powell et al., 1979; King et al., 1980,1981; Van Santen et al., 1981,1983, Cheung and Kieff, 1982; Dambaugh et al., 1984; Hennessy and Kieff, 1985). This region of the genome encodes EBNA 2, a 80- to 88-kDa nuclear protein detected by EBV-immune human sera in most cells latently infected with the virus (Dambaugh et al., 1984; Hennessy and Kieff, 1983, 1985). A number of observations have suggested that the LTl gene may have a role in initiating B cell proliferation after viral infection. For example, P3HR-1, a nonimmortalizing strain of EBV, lacks the U2 domain (Miller et al., 1974; Raab-Traub et al., 1978; Ragona et al., 1980; King et al., 1982; Bornkamm et al., 1982; Jeang and Hayward, 1983). In

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addition, transfection of human lymphocytes with a DNA fragment consisting essentially of U2 was associated with lymphocyte proliferation (Volsky et al., 1984). The LT2 gene includes the right end of U3, IR3, and the left end of U4 (Heller et al., 1982; Summers et al., 1982; Hennessy et al., 1983). This gene encodes EBNA 1, a 68- to 88-kDa protein of varying molecular mass among cells immortalized by different EBV isolates (Fischer et al., 1984; Hearing et al., 1984; Luka et al., 1984; Robert et al., 1984). Recent evidence suggests that the EBNA 1 gene may have a role in the maintenance of EBV as a plasmid in cells latently infected with the virus (Yates et al., 1984, 1985). The LT3 gene is located within U5 near TR, and unlike LTl and LT2, is highly transcribed in latently infected cells, since there are at least 60 copies of the mRNA per cell (Van Santen et al., 1981, 1983; Fennewald et al., 1984; Hennessy et al., 1984). This gene encodes a 54- to 60-kDa membrane protein referred to as LIMP. Due to its membrane location this protein may include the antigen or antigens recognized by EBV-immune T cells and may thus represent LYDMA. A third nuclear protein has been recently identified in cells latently infected with EBV and has been name EBNA 3 (Hennessy et al., 1985). It was shown that certain sera from EBV-seropositive normals and from patients with rheumatoid arthritis identify a 140-kDa protein in immunoblots of latently infected cells. This is distinct from the previously characterized EBNA 1, EBNA 2 and LIMP proteins, since specific sera to these proteins clearly failed to react with the 140-kDa protein. The EBV DNA U3 domain to the left of LT2 has been shown to encode a 4.5-kb RNA in latently infected B cells; it is therefore likely that this region (named LT4) contains the gene encoding EBNA 3 (King et al., 1980, 1982). As mentioned earlier, while most EBV-infected B cells are nonpermissively infected with the virus, a small (0-10%) proportion of these cells spontaneously replicate EBV (Sugden et al., 1979). The frequency of cells replicating the virus can be increased to 20-40% by the addition of a number of inducers, including the phorbol ester TPA (12-O-tetradecanoylphorbol-12-acetate), sodium butyrate, and corticosteroids (Gerber, 1972; zur Hausen et al., 1978; Kallin et al., 1979). Cells undergoing a productive EBV infection express the diffuse (D) and restricted (R) components of the early antigens (EA) that can be recognized by immunofluorescence with certain EBV-immune human sera (Henle et al., 1971; Pearson, 1980). Recently, monoclonal antibodies have been produced against the EBV-induced EA complex that have specificity for its D and R components (Pearson et aZ., 1983). It was determined that the EA-D specific monoclonal antibody reacts

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with two major polypeptides, with molecular weights of approximately 50-52K, whereas the anti-R monoclonal antibody precipitates a protein of approximately 85K. Recently, the genes encoding the 5052K polypeptides of EA-D were mapped to the EBV DNA BamHI M fragment (Pearson et al., 1983). In addition to EA, productively infected cells express the viral capsid antigen (VCA) in the nucleus and cytoplasm and express the membrane antigen (MA) on the cell surface. VCA and MA antigens are generally considered late viral gene products, since, unlike EA, their expression in productively infected cells can be suppressed by agents that inhibit viral DNA synthesis such as phosphonoacetic acid and acycloguanosine (Gergely et al., 1971; Nyormoi et al., 1976; Summers and Klein, 1975; Granlund and Pearson, 1977; Feigny et al., 1981). Monoclonal antibodies have been produced against two major polypeptides associated with the VCA complex. One of them recognizes a glycoprotein of approximately 125K detected both in the nucleus and in the cytoplasm of productively infected cells as well as on purified viral particles (Takada et al., 1983; Kishishita et al., 1984); the other monoclonal immunoprecipitates a polypeptide of approximately 160K detected mostly in the nuclei of EBV-infected cells and on the virion (Vroman et al., 1985). Four virus-specific proteins, gp350/300, gp220/200, p140, and gp85, have been shown to be the major components of the virus envelope; these proteins are also found on the plasma membrane of cells productively infected with EBV (Edson and Thorley-Lawson, 1981, 1983). Monoclonal antibodies have been produced to gp350/300 and gp220/ 200; interestingly, most of these monoclonals immunoprecipitate both of these glycoproteins, suggesting that they share most of the antigenic determinants (Thorley-Lawson and Geilinger, 1980; Strnad et al., 1982; Thorley-Lawson and Poodry, 1982). Recently, the genes encoding gp350/300 and gp220/200 have been mapped to the same 5kDa BamHI-L fragment of the EBV genome (Hummel and Kieff, 1982). 111. The EBV Receptor

Much evidence has been accumulated to suggest that two types of cells may naturally become infected with EBV in humans, cells of B cell lineage and epithelial cells. Since it is difficult to obtain normal human epithelial cells for experimental work, most of the information relating to EBV infection in vitro derives from studies of lymphoid cells. Cells of B cell lineage are infected with EBV in preference to

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other blood cells, and this selectivity is believed to derive from the expression of a specific cell surface receptor (Jondal and Klein, 1973). Several lines of evidence indicate that the EBV receptor on B cells is closely associated with or identical to the complement receptor type 2 (CR2) for the C3d cleavage fragment of the third component of complement (Yefenof et al., 1976; Einhorn et al., 1978, Jondal et al., 1976; Klein et al., 1978; Magrath et al., 1981; Hutt-Fletcher et al., 1983). C3 receptors and EBV receptors are generally coexpressed on B cells and B cell lines. In addition, a heteroantiserum and two distinct monoclonal antibodies to CR2, H B 5 and OKB7, have been shown to block either alone (OKB7) or with the addition of a second anti-mouse Ig antibody (HB5) the binding of purified EBV to B cells, thereby preventing viral infection of these cells (Fingeroth et al., 1984; Nemerow et al., 1985). Furthermore, Staphylococcus aureus particles to which CR2 had been adsorbed were capable of binding specifically radiolabeled virus (Fingeroth et al., 1984; Frade et al., 1985). The C3d receptor is a membrane glycoprotein of 140,000-145,OO molecular weight (Iida et al., 1983; Weis et al., 1984). Analysis of the expression of CR2 with the HB5 monoclonal antibody has revealed that among blood cells only mature B cells either resting or activated are positive, while pre B cells, immature B cells, and plasma cells are mostly negative (Tedder et al., 1984). In apparent contrast to these findings, cell lines were recently derived by EBV immortalization of fetal liver cells that were composed of “pre B cells” (Hansson et al., 1983; Katamine et al., 1984).These included cells that had their immunoglobulin genes still unrearranged and others that had intracytoplasmic p but no surface immunoglobulin. These findings indicate that immature B cells at various stages of differentiation can be infected with EBV. It is possible that the degree of CR2 expression on these cells is below the level of detection with the techniques used or alternatively that structures other than CR2 may permit EBV infection. Using an unusual EBV-transformed cell line we have recently obtained evidence for the existence of EBV binding to cell surfaces through molecules different from CR2 (Wang et al., 1985b). This B cell line was obtained by EBV infection of normal adult peripheral blood cells and was selected by limiting dilution cloning on the basis of its failure to secrete Ig. Cell surface phenotyping of this clone, named VDS-0, showed that HLA-DR, B1, and B4 antigens were expressed on over 90% of the cells, demonstrating its B cell nature. T cell and monocyte markers were absent. However, unlike the uncloned polyclonal parent cell line VDS, VDS-0 lacked receptors for

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C3d as demonstrated both by a failure to stain with three monoclonal antibodies to C3d (HB5, OKB7, and B2) and by a failure to form rosettes with C3d-coated red blood cells. In addition to these findings, V D S - 0 cells bound fluorescein-labeled EBV and absorbed out infectious viral particles (Table I). Furthermore, it was possible to superinfect V D S - 0 cells, because incubation with P3HR-1 EBV was associated with the expression of early antigens by approximately 3% of V D S - 0 cells. Thus, while there is convincing evidence that CR2 is the most common EBV receptor on B cells, structures other than CR2 may also permit EBV binding to, and infection of, certain uncommon cells. IV. B Cell Activation by EBV

After exposure to transforming EBV a proportion of the B cells express the virally encoded nuclear antigen EBNA (Reedman and Klein, 1973). This antigen, detected 24-48 hr after EBV infection, has been TABLE I SELECTED PHENOTYPIC MARKERS ON VDS-0 CELLLINE % Positive cells"

Reagent

Parent line VDS

Clone VDS-0 ~

Anti-HLA Anti-Ig (G, A, M) J5 T11 Leu M 3 B1 B2 B4 HB5 OKB7 EBV"

81 94 0.0 0.0 0.0

97 66 100 64 n.d. 64

~~

100 0.4 0.0 0.0 0.0 96 0.0

93 0.3 0.0

38

Cell surface phenotyping was performed by direct or indirect immunofluorescence. n.d., Not determined. EBV binding was determined by incubating the cells first with concentrated virus and then with the mouse monoclonal antibody 2L10 directed against the membrane antigen (gift of G. Pearson); staining of bound virus was obtained with a FITC goat anti-mouse Ig antisera.

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SYSTEM

85

defined by indirect immunofluorescence using sera of EBV-seropositive individuals (Einhorn and Ernberg, 1978; Robinson and Smith, 1981). As mentioned earlier, the EBV genome encodes at least three nuclear proteins, EBNA 1, 2, and 3, and it is likely that the nuclear staining recognized by indirect immunofluorescence of latently infected cells is due to the accumulation of these virally coded proteins. Many studies indicate that most peripheral blood B cells have EBV receptors (Fingeroth et al., 1984; Tedderet al., 1984; Nemerow et al., 1985). In addition, using radiolabeled EBV, Aman et al. (1984) have shown that the virus can penetrate most B cells. However, only a fraction of the B cells are induced by the virus to express EBNA even if nonlimiting multiplicities of infection are employed (Zerbini and Emberg, 1983; Aman et al., 1984). Purification of lymphocyte subsets has revealed that EBNA-expressing B cells derive from high density, phenotypically resting, B2-expressing B cells; low density, BB2-expressing B cells generally fail to become EBNA positive after exposure to EBV (Henderson et al., 1977; Robinson et al., 1979; Aman et al., 1984). After infection with the virus, high density B cells undergo a density shift, so that by the time EBNA is expressed they have developed into intermediate density cells (Aman et al., 1984). Approximately 48 hr after exposure to transforming EBV, B cells begin to proliferate, and 3 or 4 days later Ig secretion is detected (Bird and Britton, 1979a,b; Kirschner et al., 1979). Eventually, providing the necessary “nutrients” are present, the cells become transformed into immortal cell lines capable of continuous proliferation and associated Ig secretion (Henle et al., 1967; Pope et al., 1968; Miller et al., 1969; Gerber et al., 1969). Several lines of evidence suggest that EBVinduced proliferation, Ig secretion, and immortalization are intimately related processes and are dependent upon B cell infection with the virus (Miller et al., 1974; Bird and Britton, 1979a). It is clear that live virus is required for the induction of each of these processes and that Ig production as well as immortalization by transforming EBV occur only in EBNA-expressing B cells (Bird and Britton, 1979a; Nilsson and Klein, 1982). Limiting dilution studies have indicated that a single EBV virion is sufficient to induce EBNA expression, B cell proliferation, Ig secretion, and immortalization (Henderson et al., 1977; Yarchoan et al., 1983; Zerbini and Ernberg, 1983). In addition, the occurrence of each of these processes is independent of cooperative interactions between the EBV-infected, EBNA-expressing B cells and other lymphoid cells (Henderson et al., 1977; Bird and Britton, 1979a,b; Kirschner et al., 1979). A number of studies have attempted to quantitate the different as-

86

GIOVANNA TOSATO

pects of B cell activation by EBV in uitro. Most experiments show that only about 10-30% of the B cells succeed in expressing EBNA 48 hr after viral infection (Henderson et al., 1977; Aman et al., 1984).At this time the EBV-infected cells have not a yet begun to proliferate and therefore EBNA expression is strictly dependent on virus infection (Aya and Osato, 1974; Einhorn and Ernberg, 1978; Zerbini and Ernberg, 1983). Using limiting dilution cultures of peripheral blood T cell-depleted mononuclear cells, Yarchoan et aE. (1983) have measured the frequency of B cells capable of Ig production induced by EBV. Under the culture conditions used in these experiments only 15% of the B cells, defined as surface Ig-positive latex-noningesting cells, produce Ig in oitro after exposure to EBV (Yarchoan et al., 1983). Comparable frequencies were reported by others (MartinezMaza and Britton, 1983; Stein et al., 1983). Similar analyses have been carried out to measure the frequency of EBV-infected B cells capable of long-term proliferation in culture. Studies by Sugden and Mark showed that only about 1in 1000 peripheral blood mononuclear cells, or approximately 1 in 100 B cells, are capable of long-term growth with EBV (Sugden and Mark, 1977).We have obtained similar figures, with a mean of 2% of the B cells becoming immortalized in our cultures (Tosato et al., 1985a), while Henderson et al. (1977) have estimated that up to 10% of the T cell-depleted cord blood cells are immortalized by EBV; however, this value involved a 20-fold correction for the “inefficiency” of their culture conditions. Analysis of the relationship between EBNA expression and B cell proliferation induced by EBV has revealed that 50-95% of the EBNA expressing B cells go on to proliferate in uitro (Zerbini and Emberg, 1983). In addition, limiting dilution studies of the relationship between Ig secretion and immortalization by EBV demonstrated that only about 50% of the EBV-infected Ig secreting B cells become immortalized by the virus (Tables I1 and 111).This finding suggests that approximately 50% of the B cells infected with EBV are only transiently activated by the virus and then die (Tosato et al., 1985a). A similar analysis performed with established lymphoblastoid cell lines has demonstrated the EBV-infected B cells in long-term cell lines are generally capable of both long-term growth and associated Ig secretion (Table 111).This finding suggests that, unlike B cells freshly infected with EBV, most EBV-immortalized B cell clones are capable of continuous proliferation (Tosato et al., 1985a). It is known, however, that most EBV-transformed lymphoblastoid cell lines lose small numbers of cells at any point in time because they replicate EBV. It was reported that during a 24-hr period 1 in 104-106 cells in different

EBV AND THE IMMUNE SYSTEM

87

TABLE I1 FREQUENCIES OF B CELLSINDUCED BY EBV TO PRODUCE Ig OR TO IMMORTALIZE Experiment number"

Mean

Precursors/103 B cells Ig secretion

Immortalization

17.0 43.1 74.7 44.4 56.8

26.8 31.3 15.1 23.7

47.2

21.4

9.9

Multiple replicate cultures were established containing lo5 irradiated T cells and varying numbers of B cells preincubated with EBV; at 6 weeks, all cultures were individually tested for evidence of immortalization and Ig secretion. The precursor frequencies for immortalization and Ig secretion were calculated by Poisson statistics.

TABLE 111 CLONALANALYSISOF EBV-INDUCED Ig PRODUCTION AND IMMORTALIZATION^ Number of positive wellsb Total number of wells

IgImmor-

IgImmor+

Ig+ Immor-

Immor+

Blood #1 Blood #2 Blood #3

323 324 216

169 137 64

5 8 4

64 52 82

85 127 66

Cell line (AVM) Cell line (FMO) Cell line (RMB)

162 162 180

126 139 155

0 0 0

2 0 0

34 23 25

Source of cells

k+

a In each experiment, multiple replicate cultures were established with lo5 irradiated T cells and a small number of EBV-infected B cells obtained from either peripheral blood or an established polyclonal EBV-induced cell line. The number of B cells per well was selected to yield less than 1 precursor per well for Ig production. After 6 weeks each well was tested for Ig secretion and evidence of immortalization. b Ig-/+, Immor-/+: number of wells negative/positive for Ig production and immortalization.

88

GIOVANNA TOSATO

clonal lymphoblastoid lines release EBV virions (Sugden et al., 1979; Sugden, 1982). Taken together these studies provide a picture of the process leading to B cell immortalization (Fig. 2). Most B cells have receptors for EBV, and a high proportion can be infected with this virus; only a fraction, however, of the order of 10-30% become EBNA positive. Expression of EBNA is required for the occurrence of B cell activation by EBV but not sufficient, and only a proportion (50-97%) of the EBNA-expressing cells go on to proliferate and secrete Ig. Finally, only about 50% of the EBNA-expressing cells that have begun to proliferate and secrete Ig can sustain these activities and become immortalized. Once long-term cell lines of EBV-infected peripheral blood B cells are established, individual cell clones withing the line are generally capable of continuous cell proliferation and associated Ig production. It is known that EBV-infected peripheral blood B cells secrete all major classes of Ig in vitro (Bird and Britton, 1979a,b; Kirschner et aZ., 1979). It is also known that each class of Ig is produced by EBVinfected cells directly, without a need for cooperation by T cells and monocytes (Kirschner et al., 1979). Analysis of the precursor frequencies of B cells which are activated by EBV to secrete IgM or IgG revealed that between approximately 1: 50 and 1: 220 B cells are induced to produce IgM and only about 1: 150 to 1:450 are induced to produce IgG (Yarchoan et al., 1983). IgA-secreting precursors range EBV INFECTION

EBNA EXPRESSION

PROLIFERATION

+

IMMORTALIZATION

Ig SECRETION

E Calla

,.,

h>

t

0

. :; s8: . 'I. I-'

<_I

1.1

#: ,

I'. EBV

FIG.2. Schematic representation of B cell activation by EBV.

EBV AND

89

THE IMMUNE SYSTEM

between approximately 1:500 and 1: 1000 (Chan et al., 1986).A number of studies have addressed the question as to whether the progeny of individual B cell precursors infected with EBV can secrete Ig of multiple isotypes or whether instead they can only produce Ig of one isotype (Stein et al., 1983; Yarchoan et al., 1983; Aman et al., 1985). Using limiting dilution cultures of EBV-infected B cells, we and others found that the progeny of a single B cell precursor produced either IgG or IgA or IgM but not more than one isotype (Yarchoan et al., 1983; Tosato et al., 1985a; Chan et al., 1986).This finding suggests that EBV induces B cells to secret Ig of the isotype they have already been committed to and does not drive them to switch Ig isotype. It is interesting to note that IgM-committed B cell precursors have a significantly greater chance of generating long-term cell lines when compared to IgG- or IgM-committed B cells (Table IV) (Tosato et al., 1985a). Accordingly, IgM is the predominant Ig isotype produced by EBV-induced lymphoblastoid cell lines (Nilsson and Klein, 1982). In rare occasions, lymphoblastoid cell lines secrete specific antibody molecules, such as anti-tetanus, anti-IgC, and anti-hepatitis antibody (Steinitz et al., 1980; Shimizu et al., 1985). It is likely that these cell lines derive from immortalization of memory B cells committed to specific antibody production.

TABLE IV EBV-INDUCEDIMMORTALIZATION OF Ig CLASS-SPECIFIC B CELLPRECURSORS"

Cultures

Ig-b

Immor" Nonimmor

3 51 54

Total

IgM+ IgGIgA-

IgMIgG+ IgA-

IgMIgGIgA+

More thanone Ig class

79

25 29

3 10

3 16

22 24

132

54

13

19

46

Ig+

53

"Replicate microcultures of 25 EBV-infected peripheral blood B cells per well were estabIished with a feeder layer of lo5 autologous irradiated T cells. At the end of a 6-week culture period, the wells were scored for the presence of outgrowth and the supernatants tested for secreted IgG, IgA, and IgM. big+/-, Number of wells positivehegative for Ig secretion; IgM+/-, number of wells positivehegative for IgM; IgG+/-, number of wells positivehegative for IgG; I d + / - , number of wells positivehegative for IgA. cImmor, Wells with evidence of immortalization; Nonimmor, wells without evidence of immortalization.

90

GIOVANNA TOSATO

One would predict that if EBV infects an immature cell that has not as yet developed into a B lymphocyte, the resulting cell line would not secrete Ig. Such cell lines have been derived (Hansson et al., 1983; Katamine et al., 1984). Katamine et al. reported the establishment of a long-term EBV-transformed cell line from fetal liver that was composed of immature cells that had their Ig genes still in germline configuration, and the cells accordingly failed to express secreted and/or surface Ig (Katamine et al., 1984). Hansson et al. (1983) generated, also from fetal liver, a number of EBV-induced lymphoblastoid cell lines composed of immature B cells that contained intracytoplasmic ,u but had no surface Ig nor secreted Ig. Taken together, these observations suggest that EBV activates cells of B cell lineage out of a resting state, inducing them to proliferate and to secrete Ig if they have developed into a B cell. This virus-induced process of activation is not associated with cell maturation into a more differentiated state nor with cell acquisition of a less differentiated phenotype. Because lymphoblastoid cell lines retain many features that are characteristic of the cells from which they derive, and do not acquire new markers if not those that are directly dependent on cell activation and/or virus infection, EBV-immortalized cells are in so many respects related to B ceIls or B cell blasts induced by other activators. Numerous attempts have been made to identify serologically cell surface determinants unique to EBV-immortalized B cells. These efforts have led to the characterization of B cell activation markers which are not unique to B cells activated by EBV. These include EBVCS (also termed Blast-2, MNMG, and B532) expressed within 24 hr after activation by a variety of agents (Kintner and Sugden, 1981a,b; Rowe et al., 1982; Frisman et al., 1983; Thorley-Lawson et al., 1985) and Blast-1 expressed later, generally on day 3 (Thorley-Lawson et al., 1982). It is interesting to note that these antigens, although present to some extent on B cells activated by a variety of mitogens, are expressed at much higher levels on EBV-activated B cells (ThorleyLawson et al., 1985). There is, however, at least one cell surface protein which is unique to B cells latently infected with EBV, namely, LIMP, latent infection membrane protein (Fennewald et al., 1984; Hennessy et al., 1984). This is encoded by EBV, and its predicted plasma membrane location was confirmed by immunofluorescence using a heteroantiserum and a monoclonal antibody to the fusion protein (Hennessy et d., 1984; Mann et d., 1985). Because of its potential for permitting a virtually unlimited expansion of normal cells of B cell lineage, EBV immortalization has al-

EBV AND

THE IMMUNE SYSTEM

91

lowed the study of rare cells that could not otherwise be easily identified. For example, we have recently generated an EBV-transformed cell line from the peripheral blood of a normal adult individual that lacks secreted, surface, and intracytoplasmic Ig (Tosato et al., 1986). Phenotypically this cell line belongs to the B cell lineage, since it expresses the B cell-restricted antigens B1 and B4 while it lacks T cell- and monocyte-associated determinants. Unlike in the cell line reported by Katamine et al. (1984), both the heavy and light chain genes have undergone rearrangements. These rearrangements are nonproductive, however, because the expected heavy (y) and light ( K ) chain RNA species are not detected on Northern blotting. It could be that the Ig genes are ineffectively rearranged or, alternatively, that the heavy and light chain rearrangements are effective recombinations with the transcriptional defect representing a later abnormality. While further analysis is necessary to distinguish between these possibilities, this cell line suggests that there are cells of B cell lineage in normal adult peripheral blood with their Ig genes rearranged and yet nonproductive. As mentioned earlier, B cell activation and immortalization by EBV occur as a direct consequence of viral infection of resting B cells, only the virus and the target B cells being required, providing “standard’ culture conditions are employed. In an attempt to determine the frequencies of B cell immortalization by EBV, Henderson et al. (1977) observed that pretreatment of blood leukocytes with lipopolysaccharide results in a 300-500% increase in the number of transformation events. The mechanism involved was not clarified but appeared unrelated to stimulation of cellular DNA synthesis. In related experiments we have recently explored the role of monocytes on B cell activation by EBV and found that monocytes, unlike B cells or T cells, have a marked enhancing effect (Pike and Tosato, 1985). This phenomenon is not clearly seen when B cells are cultured at high cell densities, but when monocyte-depleted peripheral blood non-T cells are cultured at 2.5-10 X lo4 celldm1 one observes a 5- to 300-fold enhancement of EBV-induced proliferation and Ig production (Fig. 3). This is likely to derive from a larger fraction of B cells being recruited into the proliferating pool, since in the presence of monocytes the frequency of B cells induced by the virus to produce Ig and outgrow is significantly increased (Table V). High density B cells are the targets for this monocyte enhancement. In addition, since monocytes fail to influence either the rate of B cell infection with EBV or the fraction of cells expressing EBNA, monocytes appear to permit the recruitment of a higher proportion of the EBNA-infected B cells

92

GIOVANNA TOSATO

3ooo-

+ Auto1

a

2 v,

-

0)

100-

30No Additional Cells

NUMBER ( x 103) OF EBV-INFECTED MONOCYTE-DEPLETED B CELLS/CULTURE

FIG.3. Monocytes markedly enhance Ig production induced by EBV. Monocytedepleted EBV-infected B cells were cultured at varying cell densities either alone or in coculture with 50 x lo3autologous irradiated monocytes, T cells, or monocyte-depleted 3 cells. The Ig-secreting cell response was determined after 12 days in culture. Plots show geometric means of seven determinations.

into the pool of proliferating cells. Since cell-free supernatants of monocytes enhance B cell activation by EBV like the monocytes do, it is likely that these cells act through a soluble mediator. Partial characterization of this factor suggests that it is different from previously described molecules such as IL-I, IL-2, tumor necrosis factor, uromodulin, and B cell growth factor. While further studies will be necessary to clarify the molecular nature of this monocyte product, these studies demonstrate that B cell activation by EBV can be upregulated

EBV AND

93

THE IMMUNE SYSTEM

TABLE V OF EBV-INDUCED PRECURSOR FREQUENCIES Ig-SECRETING CELLS IN THE ABSENCEAND PRESENCE OF MONOCYTES PrecursodlCP B cells Experiment number"

B cells alone

1 2 3 4 5 6 7

0.2 0.2 <0.1 2.2

-

0.7

B cells + autologous T cells

B cells + autologous monocytes

0.6

17.4 13.2 11.0 32.8 11.8 52.6 16.6

-

3.8 0.2 4.0 3.3

0 Multiple replicate cultures were established containing varying numbers of EBV-infected, monocytedepleted B cells either alone or mixed with autologous irradiated T cells or monocytes. The supernatants of all wells were individually tested for the presence of Ig after 4 weeks in culture. Frequencies of EBV-induced Ig secretion were calculated by Poisson statistics.

by exogenous agents. Further support to the notion that EBV-infected B cells can be affected by stimulatory signals from the outside derives from the observation of Rossen et al. (1985),who described a soluble monocyte product of approximately 30,000 MW capable of enhancing Ig secretion but not proliferation of an established lymphoblastoid cell line. Recent evidence suggests that EBV-immortalized cells require growth factors for their continuous proliferation. These are secreted by the EBV-infected B cells themselves and can be found in the cellfree supernatant of lymphoblastoid cell lines. Gordon et al. (1984a,b) have observed that lymphoblastoid cell lines stop proliferating when seeded at low cell densities (lo4 cells/ml) and that the addition of conditioned medium obtained from lymphoblastoid cells which had been taken from their log phase was able to replace the high cell density requirement for growth. The function of the soluble activity secreted by EBV-infected cells appeared to reside in its ability to enhance the expression of transferrin receptors at the cell surface, and as a consequence permit a proliferative response to transferrin (Gordon et al., 1984b). Further study of the soluble activity produced by

94

GIOVANNA TOSATO

lymphoblastoid cells suggested that it might be similar to “T cellderived B cell growth factor,” since conditioned media of lymphoblastoid cell lines induced the proliferation of resting B cells prestimulated with nonmitogenic doses of anti-p, a function of B cell growth factor (Gordon et al., 1984a). It was also reported that B cell growth factor-containing T cell supernatants replace the high cell density requirement for growth of lymphoblastoid cell lines (Gordon et al., 1984a). Furthermore, Maize1 et al. (1983) were able to maintain in extended cultures normal preactivated B cells, which were apparently not virally infected, by the periodic addition of T cell-derived B cell growth factor. Taken together, these observations suggest that EBVimmortalized cells produce and secrete stimulatory factors for their growth. If an autocrine loop is required for the growth of EBV-transformed cells so that these cells both secrete and respond to growth factors, one can ask what the role of EBV might be in the establishment and maintenance of the immortalized state of a B cell. The virus could induce B cells to become blasts “responsive” to growth factors and could drive them to express appropriate surface receptors. These may not be unique to EBV-activated cells and could be induced by other stimuli. Indeed, the above-mentioned experiments would suggest that anti-p may act like EBV in inducing a cell to respond to B cell growth factors. In addition to inducing the expression of surface receptors, the virus might activate the infected B cells to synthesize and secrete stimulatory factors. These factors could be produced under normal circumstances by other cells and may have a role in the physiologic activation of B cells. In this model the role of EBV would be one in which the virus induces the production of growth factors and the expression of specific surface receptors for these molecules. An alternative model for the mechanism of immortalization by EBV is one in which the virus acts only inside the cell so that the EBVtransformed lymphocyte no longer requires external signals to proliferate, if not those contained in standard culture medium, clearly insufficient by themselves to maintain long-term growth. One possibility would be, for example, that virally encoded products responsible for the maintenance of viral plasmids within the transformed cell might act “in trans” on the host chromosomal DNA inducing its replication. These trans-acting molecules would at the same time be responsible for viral and cellular DNA replication. There is not sufficient evidence at this time to refute or substantiate this latter model; however, a rigorous testing of the hypothesis is presently underway using recombinant DNA techniques (Sugden et al., 1984). Yates et al. (1984) have

EBV AND T H E IMMUNE

SYSTEM

95

identified a small region of the EBV genome, the pBamC fragment composed of only 1800 bp, that acts as an origin of replication for the EBV genome; they have shown that it can function only in EBV DNApositive cell lines, suggesting that this viral origin requires virally encoded products to work (Yates et al., 1984). These viral products have been recently identified as the EBNA 1 protein (Yates et al., 1985). It is now possible to address the question as to whether this “minimal EBV replicon” can maintain cell division of a human B cell preactivated by agents other than EBV. The experiment will consist of the microinjection of the minimal EBV replicon into B cell lymphoblasts induced by anti-Ig or mitogens. If no host cell DNA replication occurs, the minimal replicon will be expanded to include other regions of the EBV DNA until cell proliferation is observed. These experiments have the potential to identify viral DNA sequences and gene products required for the maintenance of continuous B cell proliferation. V. Primary EBV Infection

Primary EBV infection in man is generally asymptomatic particularly in childhood but can be associated with a variety of clinical manifestations ranging from a mild nonspecific illness of short duration to the more severe syndrome of infectious mononucleosis (IM) (Henle and Henle, 1979a). This is characterized by malaise, sore throat, lymphadenopathy, lymphocytosis, and the appearance of atypical cells in the circulation (Niederman et al., 1968). Most of our knowledge relating to primary infection with EBV derives from studies of patients with acute EBV-induced infectious mononucleosis, and not much is known about the other manifestations of primary infection. It was suggested that the severity of the disease might be directly related to the dose of the infecting virus, but other possibilities may not be excluded, such as the mode of infection and the “status” of the host (Epstein, 1984). It is worth noting that acute I M represents a relatively unusual response to primary EBV infection. It is believed that the oropharynx is the site of initial infection with EBV. Here the virus is replicated probably in the epithelial cells of the upper respiratory tract and of the salivary glands and is then released into the oral cavity (Epstein and Achong, 1977; Lemon et al., 1977; Morgan et al., 1979; Sixbey et al., 1983, 1984). EBV infection becomes subsequently generalized with the appearance of virally infected B cells in the peripheral blood (Klein et al., 1976; Robinson et al., 1981).An additional feature of peripheral blood during acute IM is

96

GIOVANNA TOSATO

the occurrence of a prominent increase in T cells bearing a suppressor-cytotoxic phenotype. These are not virally infected and are believed to be reactive (Sheldon et al., 1973; Reinherz et al., 1980; DeWaele et al., 1981). Resolution of symptoms of the primary infection is associated with a decrease, but generally not disappearance, of both virus shedding in the oral cavity and occurrence of EBV-infected B cells in the peripheral blood (Gerber et al., 1972; Miller et al., 1973; Tosato et al., 1985b). In contrast, levels of suppressor-cytotoxic T cells return to normal values during convalescence (DeWaele et al., 1981). Primary infection with EBV is associated with the appearance of antibodies directed to the viral capsid antigen (VCA), to the early antigens (EA), and to the membrane antigen (MA), viral antigens that are known to be associated with the lytic cycle (Henle and Henle, 1979b). In contrast, the response to the nuclear antigen is delayed of a few weeks or months. The reasons for such a delay are presently unknown but may derive from EBNA not being released in an antigenic form (Klein and Klein, 1984). The epithelial cells replicating the virus probably express EBNA, but this antigen could be degraded or altered before the cells lyse. A proportion of the circulating B cells are also known to express ENBA during acute IM, but these cells may not be destroyed in a way that would make the antigen available. In this respect, it was reported that T cell immunodeficiency is accompanied by a decrease in or disappearance of the anti-EBNA response, together with an increase of anti-EANCA titers (for a review, see Henle and Henle, 197913). This would suggest that productively infected cells are a source of EA and VCA but not EBNA. It was proposed that release of antigenic EBNA may come only from latently infected circulating B cells killed by immune T cells (Klein and Klein, 1984). Among the anti-EBV antibodies produced during acute IM, antiMA antibodies are of particular interest in that they include virusneutralizing antibodies (Pearson and Qualtiere, 1978; Edson and Thorley-Lawson, 1981). As mentioned earlier, the MA antigenic complex is composed of three glycoproteins (gp350, gp220, and gp85) and one polypeptide (p140),and there is experimental evidence to suggest that gp350 and gp220 are potent inducers of neutralizing antibodies (Thorley-Lawson and Geilinger, 1980; North et al., 1982). Most of the anti-MA response during acute IM is directed to gp85 and not to gp350 or gp220 (Pearson et al., 1979), and this could explain why neutralizing antibody levels are generally low during acute IM as compared to convalescence (Rocchi et al., 1973).

EBV AND

T H E IMMUNE SYSTEM

97

Several features of primary EBV infection should be stressed. As mentioned above, a proportion of the circulating B cells are EBV infected and express EBNA (G. Klein et al., 1976). These virally infected cells are capable of generating long-term lymphoblastoid cells lines when they are transferred in vitro (Gerber et al., 1969; Chang et al., 1971; Hinuma and Katsuki, 1978; Katsuki et al., 1979). As determined by Poisson statistics of limiting dilution cultures, there are, on average, approximately 130 EBV-infected cells per lo6 circulating B cells that are capable of spontaneous outgrowth when cultured in vitro (Fig. 4)(Tosato et al., 1985a). Similar values have been reported by others (Rocchi et al., 1977). The number of EBNA-expressing B cells in the blood of patients with acute IM has been reported to be

“F

-

4L

Geo. Mean

b

3

- 1 Q

2t

+

INFECTIOUS MONO

NORMAL

FIG.4. Frequencies of EBV-infected B cells in the peripheral blood of normal EBVseropositive individuals and of patients with acute EBV-induced infectious mononucleosis. Limiting dilution cultures of peripheral blood B cells were assessed for the occurrence of outgrowth after 6 weeks, and the results were analyzed by Poisson statistics.

98

GIOVANNA TOSATO

much higher, to range between 1 and 20% of the non-T cells (Robinson et al., 1981). Since it is likely that the conditions for cell growth achieved in vivo are better than those in vitro, one can conclude that during IM there are at a minimum an average of 130 cells per lo6 circulating B cells that are capable of progressive proliferation. However, this occurs only rarely in patients with primary EBV infection, demonstrating the existence in vivo of efficient regulatory mechanisms. With the EBV-infected B cells representing a minority of the circulating cells during acute IM, the usually prominent lymphocytosis associated with this illness is composed for the most part of T cells bearing a suppressor-cytotoxic phenotype (Sheldon et al., 1973; Reinhertz et al., 1980). Studies in vitro have demonstrated that EBVactivated B cells are potent activators of autologous T cells, inducing them to proliferate and develop a cytotoxic potential. Thus, the T cell lymphocytosis during acute IM is likely to derive, at least in part, from a T cell response to EBV-induced B cell blasts (Klein and Klein, 1984). The role of serum antibody to the control of primary EBV infection appears to be limited. As mentioned above, most of the humoral response is directed toward antigens of the Iytic cycle, and of these only anti-MA antibodies have the potential to influence the course of infection because they can neutralize the virus. This may be of importance in preventing viremia and subsequent infection of circulating lymphocytes. It is not clear, however, whether neutralizing antibodies can prevent the intracellular passage of infectious virus from a productively infected epithelial cell to a susceptible B lymphocyte in contact with it, or whether they can protect epithelial cells in the oropharynx from being infected with the virus. Certainly these antibodies have no role in controlling B cells infected with EBV, since in these cells the virus is usually present in a latent form and little or no virus replication occurs. VI. Cellular Immunity during Acute EBV-Induced Infectious Mononucleosis

Much work has been devoted to the understanding of how EBVinfected B cells found in the circulation of patients with acute IM are controlled, since these cells are capable of progressively proliferating and therefore have a malignant potential. This is a fascinating area of research, and it represents the only known situation of highly efficient tumor surveillance in man amenable to experimental analysis. Two mechanisms may limit the expansion of EBV-infected B cells

EBV AND

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99

during primary infection, spontaneous death of the virally infected cells and/or their immune regulation. There is no evidence to suggest that B cells infected with EBV undergo spontaneous terminal differentiation, and, in addition, it is known that lymphocytes generally replicate little or no EBV. Thus, it is unlikely that the marked fall in the number of EBV-infected B cells after acute IM is due to the spontaneous death of these lymphocytes in uiuo. In addition; much information has been accumulated relating to the existence of a prominent cellular immune response in this illness. Two immunoregulatory mechanisms, killing and suppression, have been examined in detail. Several investigators have reported that patients with acute IM have activated cytotoxic cells in the peripheral circulation that disappear during convalescence (Svedmyr and Jondal, 1975; Turz et al., 1977; Lipinski et al., 1979; Klein et al., 1980, 1981; Patel et al., 1982). Despite much work, however, the nature and specificity of the cytotoxic response is not clear. Initial studies showed that most of the cytotoxic activity derived from Fc, receptor-negative cells and was preferentially directed toward EBV genome-positive B cells (Svedmyr and Jondal, 1975; Lipinski et al., 1979; Seeley et al., 1981). A variety of EBV-negative cell lines including K562, known to be particularly sensitive to natural killer cells, were not killed in these assays, suggesting that during acute IM a virus-specific cytotoxic response is generated. Most of the lymphoblastoid cell lines used in these assays were EBV nonproducers, and as a consequence they presumably did not express MA, the only surface determinant previously described. These findings led Klein et al. to propose that B cells latently infected with EBV express a “lymphocyte detected membrane antigen” (LYDMA) on their surface that represents the target for EBV-specific T cell killing during acute IM (E. Klein et al., 1976). Further studies of IM cytotoxic T cells showed that their killing was not restricted by the major histocompatibility complex (MHC), since MHC-incompatible targets were killed in most cases as well as autologous targets (Svedmyr and Jondal, 1975; Turz et al., 1977; Lipinski et al., 1979; Seeley et al., 1981). This lack of MHC restriction would distinguish IM virusspecific killing from most other types of antigen-specific cytotoxic cells (Zinkernagel and Doherty, 1980). More recently, however, additional studies of IM cytotoxic T cells have raised questions of their EBV specificity. Testing of many types of target cells showed that IM T lymphocytes kill EBV-negative cells as well as EBV-positive targets (E. Klein et al., 1980,1981; Patel et al., 1982). This pattern of broad spectrum cytotoxicity is commonly seen when normal peripheral blood mononuclear cells are preactivated in

100

GIOVANNA TOSATO

vitro in a variety of ways, including stimulation with antigens, mitogens, lymphokines, and, most importantly, with EBV-transformed B cells (Ortaldo et al., 1977; Seeley and Golub, 1978; Pawelec et al., 1982; Rimm et al., 1984). These observations have led some investigators to conclude that IM cytotoxic T cells are similar to “activated killer cells” triggered in vitro by various means, and it was proposed that killer cells are induced during acute IM by the circulating EBVactivated B cells (E. Klein et al., 1980; Klein and Klein, 1984). The contrasting results obtained by different studies of cytotoxic function by I M T cells are difficult to reconcile, and at this time the question relating to the true specificity of IM cytotoxic T cells is unresolved. Nonetheless, much evidence has been accumulated to conclude that during acute IM cytotoxic cells are often activated i n vivo that, in the absence of further stimulation in vitro, can kill autologous EBV-infected B cells. In addition to cytotoxic T cells, a number of investigators have reported that suppressor T cells are activated during acute EBV-induced IM (Haynes et al., 1979; Tosato et al., 1979, 1982a; Johnsen et al., 1979; Reinherz et al., 1980). These have been shown to inhibit proliferation and Ig production by normal cocultured cells. For example, as shown in Fig. 5, while normal mononuclear cells cultured alone in the presence of the T cell-dependent activator pokeweed mitogen (PWM) produce large numbers of Ig-secreting cells after 7 days in culture, these same normal cells produce very few Ig-secreting cells when cocultured with IM T cells at various ratios. No inhibition is generally observed when normal T cells are used in these coculture assays. This IM T cell suppressor function is radiosensitive and can be counteracted by the addition of a simple sugar, D-mannose and certain D-mannose derivatives (Table VI) (Tosato et al., 1983). An additional characteristic feature of IM suppressor T cells is their apparent ability to inhibit only early events of B cell activation in culture (Tosato et al., 1979). If IM T cells are added to ongoing cultures of PWM-activated mononuclear cells initiated only 24 hr earlier, no significant inhibition of the normal response was observed. In addition, since random normal mononuclear cells stimulated with PWM are inhibited in these coculture assays, IM suppressor T cells appear to be MHC unrestricted and virus nonspecific (Tosato and Blaese, 1985). The spectrum of cells inhibited by IM suppressor lymphocytes includes allogenic T cells, since it was shown that normal T cell proliferation in response to antigens and mitogens is suppressed by IM T cells. Inhibition by IM T cells does not appear to involve a soluble mediator, because culture supernatants of IM T cells do not contain

EBV AND

101

THE IMMUNE SYSTEM

10.000 -

YA UI

"

CJ

3,000

-

z

t; a

xcn

2 A

1,000-

3

m

s 0

0

z

2

E

300-

L NORMAL MNC

-

NORMAL MNC

NORMAL MNC

NORMAL T CELLS

I M T CELLS

+

+

FIG.5. Activation of suppressor T cells during acute EBV-induced infectious mononucleosis. Mononuclear cells (MNC) (0.5 x lo6) were cultured either alone in the presence of pokeweed mitogen or mixed with allogenic T cells (2.0 x lo6)from either normal individuals or patients with acute EBV-induced infectious mononucleosis. The Ig-secreting cell response was evaluated after 7 days in culture.

suppressor factors that would reproducibly mimic the inhibitory effect of IM T cells (Tosato and Blaese, 1985). It is important to note that T cell suppression during acute EBVinduced IM is not restricted to virally activated B cells but includes EBV-activated lymphocytes. This was demonstrated in cultures of IM B cells stimulated in vitro with exogenous EBV and incubated either alone or with autologous T cells (Tosato et al., 1982a, 1983). Under the conditions used, while IM B cells activated in vitro with exogenous EBV produce large numbers of Ig-secreting cells, autologous mixtures of IM B and T cells usually result in an inhibited Ig-secreting cell response. In certain TM patients, however, T cell suppression of Ig production was observed in cultures stimulated with PWM but not with EBV (Tosato and Blaese, 1985). It could be that many types of suppressor T cells are activated during acute IM with different target specificity, and that certain patients at some point of their illness have

102

GIOVASN.4 TOSATO

TABLE Vl ESHANCED ~ ~ S E C R E T CELL I N C ; RESPONSEBY SUPPRESSED CULTKWES I S THE PRESENCE OF sCC.4RS Ig-secreting cells

P\V.\l

S 11gar

Exp. 1

Esp.2

Konnal XfSC

-

Sone Sone D-Mannose a-MM

26607 9268 8932 9303

150 7620 7412 6868

honnal XlNC + Ilrl T cells

-

None Sone D-Mannose

23379 2079 5002

1720 4630

a-lllf

47.21

L-l\lannose

2203

Ciilture" ~

+ + +

+ f

+ +

370

5160 1320

Nornial mononuclear cells (\1SC, 0.5 x lo6)were cultured either alone with or without pokeweed mitogen (PWM) or with infectious inonc~nucleosis(IM) T cells (0.5 x 10'9 for 7 days. tp Sugars, urnannose, a-methyl-u-niannoside (abllf), or L-niannose, were added to the cultures at the final concentration of 25 m M . '1

suppressor T cells acting on helper T cells but not directly on B cells. Alternatively, the difference in results observed in cultures activated with either PWM or EBV is one of degree, and it could be influenced b y small variations in the experimental approach. Indeed, many types of cell-mediated immune functions have been reported to be depressed in patients with acute IM,and this could well be explained by the activation of suppressor T cells with a broad ranging inhibitory function (llangi et al., 1974; Nickoskelainen et al., 1978). Similar to the activation of cytotoxic T cells, the nature of stimuli involved in the induction of suppressor T cells during acute IM is not known. However, since exposure of normal T cells to autologous EBV-activated B cells at appropriate ratios results in the generation in uitro of suppressor T cells that are functionally iiidistinguishable from those activated during acite 1x1 (G. Tosato, unpublished results), it is possible that IM suppressor T cells are induced in cico by a similar mechanism. Killer and suppressor functions activated during acute I M share several characteristic features. Both activities are T cell mediated and lack EBV specificity and MHC restriction (Tosato and Blaese, 1985).I t could be argued that suppression is the result of IM T cells killing the proliferating and differentiating cells in coculture, rather than a dis-

EBV AND

103

THE IMMUNE SYSTEM

tinct function of IM T cells. Recently, we have addressed this issue by growing clonal T cell lines from the peripheral blood of two patients with acute EBV-induced IM, using T cell growth factor-supplemented culture medium (Wang et al., 1985a). Analysis of 152 T cell clones for their ability to inhibit PWM-induced Ig production in uitro yielded 11 highly suppressive T cell clones. When tested for killing against the natural killer-sensitive K562 cell line and against an autologous as well as an allogenic EBV-induced lymphoblastoid cell line none of these 11 suppressor T cell clones had any significant cytotoxic activity (Table VII). These findings indicate that during acute IM suppressor T cell clones are activated that have no cytotoxic function. It is interesting to note that all the suppressor T cell clones tested had the ability to delay the outgrowth of autologous B cells infected in uitro with exogenous EBV, suggesting that suppressor T cells alone play a role in the control of EBV-infected B cells. Killer and suppressor functions displayed by IM T cells have the potential to influence the course of EBV infection in uivo. Cytotoxic cells would destroy EBV-infected cells, while suppressor T cells would limit their growth by exerting an antiproliferative effect. In apparent contrast to this potential for effective immunoregulation, at

TABLE VII AND CYTOTOXIC ACTIVITYOF SELECTEDT CELLCLONES FROM SUPPRESSOR PATIENTS WITH ACUTE INFECTIOUS MONONUCLEOSIS

% Suppression of normal Ig productiona

Cytotoxicity (% lysis)b, targets

Clone

PWM induced

EBV induced

Autologous EBV B line

Allogeneic EBV B line

K562

06 18 24 50 56 68

77 86 95 69 79 75

65 9 -11 64 90 72

0.2 1.2 -1.3 - 1.6 -0.8 0.0

4.4 -2.3 -7.0 2.3 -9.3 -6.6

1 .o 2.7 -0.5 - 1.0 -3.2 - 1.2

~~

Suppression of normal Ig production was tested in cultures of 124 x 103 normal mononuclear cells cultured in the presence of pokeweed mitogen (PWM)either alone or with 1 x lo5cloned infectious mononucleosis T cells. EBV-induced cultures were performed with 50 x lo6B cells and 25 x 103 cloned T cells. b Cytotoxicity was determined at a target : effector ratio of 1 : 100.

104

GIOVANNA TOSATO

least in uitro IM T cells have consistently proven to be ineffective in controlling the outgrowth of autologous B cells endogenously or exogenously infected with EBV (Rickinson et al., 1980a; Schooley et al., 1981).It has long been known that IM mononuclear cell cultures give rise to spontaneously outgrowing B cell lines despite the presence of T cells (Pope, 1967). In addition, when we tested the possibility that IM T cells might at least reduce the frequency of autologous EBVinfected B cells capable of spontaneous outgrowth in uitro, we found that this was not the case (Tosato and Blaese, 1985). Whether IM B cells were cultured alone or in the presence of autologous IM T cells, the frequencies of spontaneous immortalization were the same. These findings could be interpreted to suggest that IM T cells represent an ineffective control against the B cells naturally infected with EBV. However, since there is so much evidence to indicate that, at least in short-term assay systems, IM T cells mediate a number of regulatory functions including killing and suppression of EBV-infected B cells, we have suggested that long-term outgrowth assays may not provide an adequate testing of IM T cell function (Tosato and Blaese, 1985).In this respect it is worth mentioning that IM T cells, unlike T cells from normal individuals, have a very short life in uitro under conventional culture conditions, with the majority of cells dying within 24 hr (Moss et al., 1985).It is possible that peripheral blood T cells during acute IM are terminally differentiated cells and short-lived both in oiuo and in uitro. In vivo they would be continuously replaced with functionally active T cells. Alternatively, since IM T cells can be propagated in culture for a prolonged period of time in medium supplemented with T cell growth factor, they may not be short-lived itt uiuo, and standard culture conditions may cause their premature death. Either of these alternatives would explain why IM T cells may not display their potential for control of EBV-infected B cells in long-term assays requiring 4-6 weeks of incubation. An alternative, and perhaps provocative, view of the T cell response observed during acute IM is that it may not reflect the activation of regulatory cells capable of limiting in uiuo the expansion of EBVinfected B cells but rather may contribute to the disease process. It was observed that many of the patients with the most severe clinical manifestations of 1M are also those displaying the greatest T cell lymphocytosis. In these cases, rather than reflecting the magnitude of the immune response to a particularly great load of virally infected cells, the T cell lymphoproliferation might involve activation of mostly nonfunctional clones irrelevant to the control of the infection and contributing to the disease process. In this view, only a minor component of

EBV AND

THE IMMUNE SYSTEM

105

the IM T cell response on the whole would include suppressor and cytototoxic functions effective against EBV-infected cells. This reactive T cell response would also include T cells capable of preventing the generation of more effective and specific types of immune responses. VII. Chronic Asymptomatic EBV Infection

Primary EBV infection whether asymptomatic or associated with

IM is followed by the establishment of a virus carrier state that continues for years and probably for the lifetime of the host. Between 10 and 60% of EBV-seropositive normal individuals shed infectious virus in pharyngeal secretions at any one time (Gerber et al., 1972; Miller et al., 1973).Virus present in this site is generally capable of inducing cord blood cells to immortalize. In addition, under the appropriate culture conditions, bloods from virtually all EBV-seropositive normal individuals give rise spontaneously, without the addition of exogenous virus, to long-term lymphoblastoid cell lines that have a B cell phenotype and express EBNA (Gerber et al., 1969; Chang et al., 1971; Nilsson, 1979). The presence of virally infected B cells in the circulation of EBV-seropositive normals cannot be determined by EBNA staining of peripheral blood B cells, because the frequency of EBVinfected cells is quite low. Using limiting dilution cultures of peripheral blood non-T cells, we have calculated that, on the average, approximately 3 in lo6 circulating B cells are capable of “spontaneous outgrowth” when transferred in uitro (Tosato et al., 1984a, 1985b). This would suggest that, at a minimum, 3 in lo6 peripheral blood B cells are infected with the virus in EBV-seropositive normals. It is important to point out that the EBV carrier state is characterized by virus persistence in the same sites in which it can be found during primary infection. Also, the type of infection in these sites, productive in the oropharynx and mostly latent in peripheral blood, is the same in IM patients and in asymptomatic carriers. The difference appears to be only quantitative, with a greater viral burden being found during primary infection. In EBV carriers, the relationship between lytic EBV infection in the upper respiratory tract and latent infection of the circulating B cells is not clear. Moss et al. (1981b) have proposed that, similar to what happens during primary infection, the virus released in the oropharynx continuously goes to infect B cells which in turn reach the peripheral blood. Here the virally infected cells would be continuously killed by specific cytotoxic T cells. In this view, the pool of circulating

106

CIOVANNA TOSATO

EBV-infected B cells would be ever changing and composed of newly infected and short-lived B cells that have not as yet become the targets for specific killer T cells (Moss et al., 1981b). A very precise balance between virus replication, B cell infection, and immune T cell killing of virally infected cells would assure that the number of EBV-infected circulating B cells be roughly the same at any time point. Indeed, the frequencies of EBV-infected B cells in peripheral blood of a given normal EBV-seropositive individual seem to range within relatively narrow limits over time (1-10 in lo6 B cells) (G. Tosato, unpublished results). In support of the view that in EBV-seropositive normals a continuous low grade replication of the virus in the oropharynx is responsible for the regular input of EBV-infected B cells in peripheral blood, Yao et al. (1985) reported that a direct relationship appears to exist between the level of virus shedding in the throat and the number of virus-infected B cells in the blood. This direct relationship could not be confirmed in two donors with detectable numbers of EBV-infected cells in the circulation who consistently failed to show virus shedding on multiple testing. In somewhat similar experiments we have measured the number of circulating spontaneously transforming B cells in a group of individuals treated with intravenous acyclovir at the dose of 1500 mg/m2 (G. Tosato and S. E. Straus, unpublished). In uitro acyclovir inhibits lytic EBV infection while it does not affect EBV latency (Colby et al., 1980; Pagano and Datta, 1982),and in uivo it has been reported to eliminate or reduce EBV shedding in the saliva (Pagano et al., 1983). In contrast to the results reported by Yao et aE., we found that acyclovir treatment was not accompanied or followed by a significant variation in the number of circulating EBV-infected B cells capable of spontaneous immortalization when transferred in uitro. Similar results were reported by others (Pagano et al., 1983). These findings would suggest that EBV infection in the peripheral blood is not directly dependent upon virus input from the oropharynx. An alternative model of virus persistence in EBV-seropositive normals was proposed by others (Crawford et al., 1978; Klein, 1980; Tosato and Blaese, 1985). In this view, the EBV-infected circulating B cells would be long-lived and proliferating, perhaps, progeny of cells originally infected with the virus at the time of primary infection. The overall size of the virus-infected circulating B cell population would be controlled by the host’s cellular immune mechanisms. These regulatory controls would prevent the expansion of EBV-infected cells. In addition, similar to what happens to “normal” lymphoblastoid cell lines cultured in uitro, a small proportion of the EBV-infected B cells

EBV AND THE IMMUNE

SYSTEM

107

would replicate the virus at any one time, and this would provide a source for late viral antigens. Support for this model derives from the observation that only a fraction of EBV-seropositive normals (at best 60%) shed transforming virus at any time point, while they regularly harbor EBV-infected cells in the peripheral blood (Gerber et al., 1972; Miller et al., 1973). In addition, the presence of neutralizing antibodies might prevent B cells from being infected with virus produced in the oropharynx. While further, more direct evidence is necessary to establish how the life-long EBV infection of circulating B cells is maintained, it is conceivable that different mechanisms may contribute to it; the chronic or intermittent lytic infection occurring in the oropharynx may be responsible for some B cells becoming newly infected with EBV at certain time points, while some B cells originally infected with the virus, or their progeny, may be long-lived. In addition to the presence of virally infected B cells in the circulation and intermittent virus shedding in the oropharynx, the chronic carrier state is associated with a characteristic pattern of serum immunity to the virus (Henle and Henle, 1979b). This includes antibodies to VCA, EBNA, and MA. Antibodies to EA are usually undetectable or present at low levels often at the threshold of detectability. Unlike patients with acute IM, EBV-seropositive normals have only IgG antibody to VCA, and the response to MA includes antibody to gp350/220. Among the anti-EBV antibodies produced after primary infection, only anti-MA antibodies to gp350/220 have the potential to influence the virus carrier state because they are neutralizing and, in addition, can mediate antibody-dependent cellular cytotoxicity (Jondal, 1976; Pearson and Orr, 1976; Qualtiere et al., 1982). The latter regulatory control for MA-expressing B cells has been shown not to be operational during acute IM, because no significant response to gp350/220 is usually present at this time. The continuous presence of specific antibody to late viral antigens after primary infection suggests that EBV is replicated either continuously or frequently in some site, either the oropharynx or peripheral blood. VIII. Cellular Immunity during Chronic Asymptomatic EBV Infection

Several lines of evidence suggest that after primary infection T cell immunity is responsible for the regulation of B cells latently infected with EBV. If peripheral blood B cells from EBV-seropositive normals are cultured in vitro, without the addition of exogenous virus, spontaneous immortalization of EBNA-expressing B cells is regularly observed (Chang et al., 1971; Nilsson, 1971; Gerber, 1972; Tosato et al.,

108

GIOVANNA TOSATO

1984,1985b). However, if these B cells are cocultured with autologous T cells at appropriate ratios, no immortalization occurs. This inhibitory effect is T cell specific, not being observed when other types of cells are added. Similarly, if mononuclear celIs from EBV-seropositive individuals are placed in culture, no spontaneous immortalization is generally observed unless T cells are functionally inactivated by the addition of cyclosporin A or an anti-T cell receptor antibody (Tosat0 et al., 1982b). Progressive expansion of EBV-infected B cells rarely occurs in uiuo. It has been described, however, in patients treated either with high dose cyclosporin A or with an anti-T cell receptor antibody, suggesting that T cell immunity is important for the control of EBV-infected B cells in uiuo (Calne et aZ., 1979; Thiru et aZ., 1981; Martin et al., 1984). In addition to their ability to regulate the spontaneous outgrowth of B cells naturally infected with EBV, T cells have been shown to inhibit markedly the immortalization of autologous B cells newly infected with EBV in vitro. This phenomenon was originally described by Thorley-Lawson et al. (1977). In these experiments, B cell-enriched populations of normal adult peripheral blood were exogenously infected with EBV in vitro and cultured with or without the addition of autologous T cell-enriched populations. Microscopic examination was used to determine the time of outgrowth of EBV-immortalized B cells in these cultures. It was observed that the addition of increasing concentrations of autologous T cells resulted in a delay of the time of outgrowth of EBV-infected cells. This result could not be explained as an artifact due to poor culture conditions present in the T cell-containing cultures, since newborn T cells failed to delay the EBV-induced B cell growth. In further studies of this phenomenon, it was demonstrated that only T lymphocytes from EBV-immune individuals can inhibit the immortalization of autologous B cells exogenously infected with EBV in vitro (Moss et al., 1978,1979; Rickinson et al., 1979,1980a). When mononuclear cells from adult EBV-seronegative individuals and from newborn bloods were infected with EBV in uitro, foci of EBNA-positive cells appear within 8-10 days postinfection, and these foci progressively expand, giving rise to lymphoblastoid cell lines. Similar cultures established with mononuclear cells from EBV-seropositive individuals initially proliferated during the first 8-10 days in culture; however, the cells were then observed to die, and no immortalization followed. T cell inhibition of B cells either naturally infected with EBV in uiuo or experimentally infected with exogenous virus in vitro are believed to reflect, at least in part, similar activities of the T cells. In the

EBV AND

THE IMMUNE SYSTEM

109

presence of exogenous EBV, a large proportion of the B cells becomes infected with the virus and has the potential for immortalization; at the same time specific memory T cell subsets within the T cell pool become selectively activated and expand. Because experimental B cell infection with exogenous virus permits the expansion of a regulatory T cell subset that most likely represents a minor cell population in vivo, the general approach has been to examine the cellular responses that can be elicited by stimulation with B cells infected in vitro with EBV. Using this experimental system, a variety of cellular regulatory mechanisms have been shown to become activated against EBV-infected B cells and to contribute to their control. The best characterized function of EBV-immune T cells is cytotoxicity, described mostly in the extensive work by Rickinson et al. (for a review, see Rickinson and Moss, 1983). These investigators have demonstrated that T cells capable of inhibiting the outgrowth of autologous EBV-infected B cells have the capacity to kill these B cells, as demonstrated by chromium-release assays (Misko et al., 1980; Moss et al., 1981). Early results had shown that the cytotoxic response was not induced by the virus but by virally infected cells, because it could be elicited by exposure of the T cells to the autologous B lymphocytes from the EBV-transformed lymphoblastoid cell line (Rickinson et al., 1979; Misko et al., 1982; Wallace et al., 1982b). In addition, it was reported that the cytotoxic cells are characterized by their EBV specificity, HLA restriction, and suppressor-cytotoxic phenotype (Misko et al., 1980; Rickinson et al., 1980b; Wallace et al., 1981,1982a,b; Moss et al., 1981a). These features can best be appreciated when the T cells are precultured for 10 days with autologous EBV-transformed cells at a relatively high responder to stimulator ratio (40 : 1).When activated in this manner, the T cell populations have been reported to show strong reactivity against the autologous EBV-transformed cells and not to kill any EBV-negative targets, including autologous mitogenstimulated cells and natural killer-sensitive cell lines (Misko et al., 1980; Moss et al., 1981a; Wallace et al., 1982~). It was therefore concluded that the cytotoxic response elicited in these cultures is directed toward determinants induced by EBV and expressed by B cells latently infected with the virus. The existence of such a determinant, LYDMA, had been previously proposed to explain the EBV-selective T cell response during acute IM (Moss et at., 1981b). An important and still unresolved issue is the molecular identify of LYDMA. Many investigators have attempted to raise monoclonal antibodies to EBV-transformed cell surface determinants with specificity for virally infected blasts, but these efforts have

110

GIOVANNA TOSATO

been unsuccessful. To date, no antibody has recognized a virally induced surface membrane component in latently infected B cells that would interfere with EBV-specific T cell recognition. The existence of a membrane protein that is encoded by EBV and expressed in B cells latently infected with the virus, however, has recently been demonstrated and has been called LIMP, latent infection membrane protein (Fennewald et al., 1984; Hennessy et al., 1984). Its molecular weight is approximately 60,000, and the characteristics of the predicted primary amino acid sequence include a hydrophilic amino terminus, six markedly hydrophobic 20-amino acid segments separated from each other by five less hydrophobic 8-amino acid segments, and a 210-amino acid hydrophilic and acidic carboxyterminal domain (Fennewald et al., 1984). The location of LIMP with respect to the plasma membrane is not known with certainty, but it appears that the amino- and carboxy-terminal domains of the protein are in the inner aspect of the membrane (Fennewald et al., 1984; Mann et al., 1985). In addition, since the first reverse turn of the protein, unlike the second and the third, is susceptible to protease cleavage in live cells, it is likely that it is exposed on the outer surface (Liebowitz et al., 1986).Whether or not this small domain, comprising probably less than 10 amino acids, is recognized by immune T cells still remains to be seen. Use of selected polypeptides, specific antibodies, and cells transfected with the LIMP gene will certainly help to determine whether LIMP is LYDMA. An alternative possibility is that insertion of the EBV protein into the membrane may alter the tridimensional conformation of adjacent proteins, thus generating a new antigenic determinant. As mentioned above, Rickinson et al. (1980b) have reported that EBV-specific cytotoxic cells are MHC restricted. Use of panels of allogenic targets with known histocompatibility antigens as well as blocking experiments with monoclonal antibodies have suggested that class I HLA antigens are the important restricting elements (Rickinson et al., 1980b; Misko et al., 1980; Wallace et al., 1981,1982~).The effector cells of EBV-specific cytotoxicity have been shown to express a supIt is still unclear, pressor-cytotoxic phenotype (Wallace et al., 1982~). however, whether helper T cells have a role in the in vitro induction of the cytotoxic response; on this issue contrasting results have been reported (Tsoukas et al., 1982; Konttinen et al., 1985). Analysis of virus-specific cytotoxic T cell function has been extended with the generation of T cell clones (Wallace et al., 1982a; Meurer et al., 1983; Slovin et al., 1983; Misko et al., 1984). With this new tool the presence of cytotoxic T cells specific for EBV-infected B

EBV AND THE

IMMUNE SYSTEM

111

cells has been generally confirmed. Most of these clones have been reported to express a suppressor-cytotoxic phenotype and to be restricted by class I MHC. Occasionally, however, virus-specific cytotoxic T cell clones have been isolated that had a helper phenotype and demonstrated a restriction for class I1 MHC (Meurer et al., 1983; Misko et al., 1984). In one study the restriction elements for some of the clones were difficult to assess (Tanaka et al., 1982). Evidence has also been obtained for the existence of a suppressor, noncytotoxic, T cell regulation that has features of EBV specificity. The assay system used to assess this regulatory function has been called “late suppression” (Tosato et al., 1981, 1982a,b). Here activation and inhibition of virally infected B cells is monitored by counting the number of Ig-secreting cells produced by the EBV-infected B cells, rather than by a visual assessment of immortalization or measure of lymphocyte proliferation. Because of its quantitative nature and its ability to reflect only the status of the EBV-infected Ig-secreting cells, this system has a number of advantages over the previously employed methods. Using this assay, EBV-infected B cells from either EBVseropositive or EBV-seronegative donors produce increasing numbers of Ig-secreting cells, reflecting the occurrence of B cell activation by the virus. A marked inhibition of the B cell response is observed if autologous EBV-seropositive T cells are added at physiologic ratios. This inhibition is not observed if the autologous T cells are from a seronegative individual. The kinetics of the inhibition are characteristic, with the EBV immune T cells showing no effect on the EBVinduced Ig response during the first 8-10 days in culture and exerting prominent suppression after this time (Fig. 6). This inhibitory effect is not observed if the virus immune T cells are irradiated or if appropriate concentrations of cyclosporin A or anti-P19 monoclonal antibodies are added to the cultures (Tosato et al., 1982b). This suggests that in uitro proliferation as well as antigen-specific recognition are required. Among lymphocyte subsets, T cells expressing a suppressor-cytotoxic phenotype mediate late-acting suppression and can exert this function alone without need of cooperation by T cells with a helper phenotype (Table VIII) (Tosato et al., 1985~).In addition, natural killer cells do not appear to play a role in this virus-specific system, since depletion of these cells does not change the degree or kinetics of suppression. A characteristic feature of late-acting suppression is that the EBVseropositive T cells can be added to the autologous EBV-infected B cells up to 3-4 days after culture initiation to cause suppression, but not at a later point in time (Tosato et al., 1982a).This finding suggests

112

GIOVANNA TOSATO

DAYS

FIG.6. Typical experiment showing that T cells from a normal EBV-seropositive individual have a delayed suppressor activity on EBV-induced Ig production by autologous B cells. EBV-infected B cells (0.5 x lo6)were cultured either alone ( 0 )or in the presence of autologous unirradiated (A) or irradiated (3000 R) (m) T cells (2.0 x lo6). The Ig-secreting cell response was determined at the indicated time points.

TABLE VIII T CELLSWITH SUPPRESSOR-CYTOTOXIC PHENOTYPE MEDIATELATE-ACTING SUPPRESSION Ig secreting cellsb

Cell cultures"

Exp. 1

Exp. 2

Exp. 3

B cells B + T cells B + T8 cells B + T4 cells

34,175 3,100 1,588 22,372

34,400 5,210 7,900 25,800

33,948 7,781 3,501 22,114

B cells (20 x 103) were cultured either alone in the presence of EBV or mixed with autologous EBV-seropositive T cells (100 x lo3). The Ig-secreting cell response was determined after 14 days of culture.

EBV AND THE

IMMUNE SYSTEM

113

either that the EBV-infected B cells express only the appropriate stimuli for induction of suppression during the first 3 days in culture or alternatively that the B cells can be suppressed only during the initial 3 days of culture, even though the effects of such inhibition can be appreciated at a later time. Since cytotoxic T cells can be induced by EBV-immortalized B cells and can kill these same cells, it would appear that a noncytotoxic process is involved in the regulation of EBV-infected B cells as it is appreciated in late suppression assays (Tosato and Blaese, 1985). Further evidence for the involvement of a noncytotoxic pathway is the observation that if the T cells are removed from the suppressed cultures the residual B cells infected with EBV are capable of rapidly differentiating into Ig-secreting cells (Tosato et al., 1982a). This reversibility suggests that T cell regulation does not occur exclusively through a cytotoxic mechanism and that some of the B cells are suppressed but not killed. In addition, we have recently been able to grow a continuous T cell line from a seropositive normal individual that has no detectable cytotoxic activity and, at the same time, can profoundly inhibit B cell activation by the virus (G. Tosato, unpublished results). Recent experiments have demonstrated that interferon inhibits EBV-induced proliferation and Ig production in vitro (Anderson et al., 1983, 1984; Hasler et al., 1983; Lotz et al., 1985). Unlike the situation with a- and p-interferons, EBV-infected B cells are susceptible to suppression mediated by y-interferon up to 3-4 days after virus infection but not at a later time (Lob et al., 1985). In addition, EBVinfected mononuclear cell cultures have been reported to produce yinterferon in vitro, with maximal secretion occurring 5-7 days after virus infection (Lotz et al., 1985). Also, T cells are believed to be a source for y-interferon production. Taken together, these results suggest that y-interferon may have an important role as a mediator for inhibition of EBV-induced B cell activation as it is appreciated in “ late suppression” assays. Since late suppression is mediated only by EBV-immune T cells, presumably it involves specific memory T cells that become activated in vitro upon recognition of a virus-induced determinant exposed on autologous B cells. Such a determinant has the characteristics attributed to LYDMA. If indeed the suppressor effector phase is mediated by a molecule such as y-interferon, the virus specificity of suppression would be determined only by a specific induction. Unlike cytoxicity, where both the induction and the effector pathways are believed to be

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specific, the effector phase of suppression could be virus nonspecific. The concept that an effective and specific control mechanism for EBV-infected B cells might occur without eliminating the infected cells has been previously proposed. It was observed that under appropriate conditions, adult EBV-seropositive, but not EBV-seronegative, mononuclear cell cultures infected with EBV failed to give rise to immortal cell lines if they were cultured on a feeder layer of human fibroblasts, but they did immortalize if no feeder layer was added (Moss et al., 1976, 1977). In the presence of the fibroblasts the B cells infected with EBV, and expressing EBNA, would not proliferate but would remain alive for a prolonged period. If the fibroblasts were removed after 1 month of culture, the EBV-infected B cells would rapidly give rise to immortal cell lines. The regulatory controls involved in this system have not been identified, mainly because of the variability of results. However, this pattern of growth inhibition suggests that the fibroblasts exert or induce a noncytotoxic reversible control of the EBV-infected cells. In addition to the presence of regulatory mechanisms that are found only in EBV-seropositive individuals, a number of investigators have reported that virus-nonspecific immunity may contribute to the control of EBV-infected cells in vitro. The existence of such nonspecific regulation was suggested by the observation that T cells derived from either adult EBV-seropositive or EBV-seronegative individuals demonstrate an ability to delay, although not to prevent, the growth of autologous B cells infected in vitro with EBV (Shope and Kaplan, 1979; Bardwick et al., 1980; Masucci et al., 1983). Newborn T cells demonstrate no activity in this system. Analysis of T cell subsets mediating this nonspecific regulation has revealed that most of the activity derives from low density T lymphocytes that bear Fc, receptors, characteristics which have been attributed to large granular lymphocytes with natural killer function. These T cells do not need to proliferate in culture to mediate their effect and they appear to act within the first few hours, because neither irradiation nor their early removal from the culture seem to interfere with their inhibitory function that is measured several days later (Thorley-Lawson, 1980; Depper et al., 1981). Masucci et al. (1983) have proposed that this virus-nonspecific regulation could be mediated by a cytotoxic activity, because the cells that are involved are large granular lymphocytes capable of natural killer activity. In support of this possibility, it was reported that T cells exposed to autologous EBV-transformed B cells in vitro, particularly if cultured at low responder to stimulate ratios, do kill the natural killer-

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sensitive cell line K562, in addition to killing the autologous line (A. B. Rickinson, personal communication). Thorley-Lawson (1980) has proposed that a noncytotoxic antiproliferative effect mediated by a soluble factor might contribute to the nonspecific T cell regulation of EBV-infected B cells. This factor was believed to be a-interferon because suppression was abrogated by heteroantisera raised against leukocyte interferon-containing material and was mimicked by preparations containing human leukocyte interferon (Thorley-Lawson, 1981). More recent studies on the role of interferons in the regulation of EBV-infected B cells have confirmed that a-interferon, in this case a recombinant reagent, reduces EBVinduced proliferation and Ig production (Garner et al., 1984; Lotz et al., 1985). Time kinetics experiments suggested that a-interferon had to be added at the time EBV infection or soon thereafter to cause inhibition. In addition, a-interferon appeared to act directly on the virus-infected B cells, without contribution from macrophages or natural killer cells (Lotz et al., 1985).While it is possible that a-interferon is the mediator, or one of the mediators, of virus-nonspecific inhibition of EBV-infected B cells, further studies are needed to show whether inhibitory concentrations of such a molecule are physiologically produced in the inhibited cultures and whether or not T cells can either produce it or induce its production. Also, recent experiments have shown the p-interferon when added to EBV-infected B cells is also inhibitory and its mode of action and effects could not be distinguished from those of a-interferon (Lotz et al., 1985). Use of EBV to infect B cells in vitro has permitted one to unveil distinct regulatory mechanisms elicited by the virally infected cells. These include specific cytotoxicity, specific suppression, natural killer activity, and nonspecific suppression possibly mediated by ainterferon. Since EBV-infected cells have both in vitro and in vivo the potential for unlimited growth it is hardly surprising that a variety of regulatory defenses may be involved in their control. While it is likely that the different regulatory processes demonstrated in vitro are also activated in vivo, very little is known about their relative role and contribution to the control of natural infection with EBV. One can predict that each regulatory process has a precise role in preventing the growth of EBV-infected cells present in vivo. It is hoped that with more information gathered about the molecular events involved in the activation of each pathway and on the sensitivity of the virally infected B cells to different types of inhibitory signals, we shall appreciate how this oncogenic herpesvirus is so successfully controlled.

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IX. Concluding Remarks

Epstein-Barr virus is a ubiquitous and potentially oncogenic pathogen for man. Once infected with the virus, the B cell acquires the potential to grow indefinitely. Tumor development, however, rarely occurs in the EBV-infected individual because strict controls assure that the virus reaches a state of equilibrium with its host. At least some of these controls are immunologic, and they act at several levels to suppress the growth of virally infected cells. Part of the uniqueness of the interaction between EBV and the immune system arises from the fact that the target for infection is the immune system itself. This imposes certain controls on the infected cells that derive from the multitude of regulatory pathways that involve the B cell as a central element of the immune system. Much information has been accumulated relating to the interactions among cells of the immune system, and this knowledge has helped us immensely to unravel the defense mechanisms against EBV. During primary infection, nonspecific suppressor and cytotoxic T cells that become activated in uiuo regulate and destroy EBV-infected B cells. After primary infection, a few B cells continue to harbor EBV in a latent form, and, as a consequence, are capable of unlimited proliferation. At this time, in conjunction with nonspecific functions, virus-specific suppressor and cytotoxic T cells that become activated continuously act to limit the expansion of EBV-infected cells. I n addition to teaching us about regulatory controls that prevent malignant B cell proliferation, EBV has just begun to be especially useful for the understanding of B cell growth. The virus immortalizes B lymphocytes, providing or inducing all the signals that are necessary and sufficient to maintain long-term growth in culture medium. This has permitted us, for example, to appreciate the importance of transferrin and B cell growth factor. It is certainly clear from this article that many questions remain unanswered concerning how a B cell becomes immortalized by EBV and how the immune system deals so successfully with a transforming virus. It is very important that this system be more completely understood, since it represents the only known situation of a highly efficient tumor surveillance in man that is at present amenable to experimental analysis. REFERENCES Adams, A., Lindahl, T., and Klein, G . (1973). Proc. Natl. Acad. Sci. U.S.A. 701, 28882892.

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THE USE OF CELL MARKERS IN THE STUDY OF HUMAN HEMATOPOIETIC NEOPLASIA Wendy H. Raskind and Philip J. Fialkow Department of Medicine, University of Washington, Seattle. Washington 98195

I. Introduction

This article summarizes the major findings that have led to current understanding of the clonal development and progression of human hemopoietic neoplasia. Determination of whether a cellular proliferation is monoclonal or polyclonal has important pathogenetic implications. Polyclonal proliferations are often the result of normal processes in which a tissue responds appropriately to an exogenous stimulus. Examples of such “normal” expansions include granulocytosis induced by bacterial infection, lymphocytosis in response to viral infection, and erythrocytosis secondary to hypoxemia. Monoclonal proliferations, on the other hand, reflect disordered hemopoiesis in which a clone of cells gains in uiuo proliferative advantage. This may occur, for example, through mutations that result in alterations such as escape from normal cell cycle control, extended proliferative life span, or loss of dependence on growth factors. Therefore, the finding that a cell population is monoclonal suggests that it is neoplastic. The ability to detect monoclonality in proliferating cells requires a marker system that enables the progeny of different cells to be recognized. Markers that have been used effectively to investigate human lymphoid neoplasia include immunoglobulin (Ig) proteins synthesized by relatively mature cells of B-lymphoid origin, DNA sequences coding for these Igs, and T-cell receptor gene rearrangements. For diseases of lymphoid and nonlymphoid origin the mosaicism created by X-chromosome inactivation in females has been exploited. For the latter studies, the X-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) is a particularly useful marker, and recently X-linked restriction fragment length polymorphisms (RFLPs) have been used. Karyotypic differences are also useful for tracing cell populations. Beyond determining whether a disease state is clonal at the time of presentation, studies with cell markers allow characterization of the 127 ADVANCES IN CANCER RESEAHCH, VOL. 49

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clonal state throughout subsequent stages of the disease and definition of the differentiative expression of the stem cell involved by the clonal proliferation. Five major conclusions have been reached from studies of hemopoietic neoplasms:

1. Human hemopoietic neoplasms develop clonally. 2. Human leukemias, along with other neoplasms, progress through a series of steps by subclonal expansion. 3. Chronic lymphoid neoplasms involve progenitors in which differentiative expression is apparently restricted to the lymphoid pathway. 4. Chronic myelocytic leukemia and other chronic myeloproliferative disorders involve stem cells pluripotent for myeloid and lymphoid lineages. 5. Acute myeloid leukemias are a heterogenous group of related diseases: In some patients the involved stem cells show pluripotent differentiative expression, whereas in others largely unipotent differentiative expression is found. It should be emphasized, however, that clonality detected by genetic marker studies pertains only to the stage at which the disease is studied. It is possible that the neoplasm had origin in many cells, but by the time disease is clinically overt only one clone is evident, i.e., clonal development.

II. Marker Systems

A. SOMATICCELLGENEREARRANGEMENTS IN LYMPHOID CELLS

1 . Immunoglobulins (Igs) In the case of B-lymphoid proliferations the process of B-cell maturation itself provides several markers for tracing lineage and clonality in disease states. Each mature B lymphocyte expresses a single light chain, K or A. A normal polyclonal population of B lymphocytes is comprised of a mixture of cells producing these light chain species. In a disease state, the detection of a sing1,elight chain type is consistent with, but not proof of, clonal proliferation. The Igs were the first cell markers used to suggest clonality in a neoplastic disease-multiple myeloma (Martensson, 1963). Through a complex process of DNA rearrangement and somatic mutation a functional antibody gene is produced in developing B

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lymphocytes (reviewed in Tonegawa, 1983). One portion of each antibody created in this way is unique (the variable region of the molecule), and only progeny of the same parent cell will express antibodies with the same variable region. The configuration of the antibody, influenced by its variable region, is itself an antigen, called an idiotype (reviewed in Geha, 1981). Given the vast potential for antibody diversity, with the myriad antibodies produced by a normal polyclonal Bcell population, the ability to ascribe a specific idiotype to a population of B cells signifies clonal expansion.

2. Immunoglobulin Gene Loci The discovery of restriction endonucleases led to development of techniques for detecting sequence differences between DNAs of various sources (reviewed in Nathans and Smith, 1975). Restriction endonucleases have the ability to cleave DNA at sites of specific nucleotide sequences. The DNA segments are then size-separated by electrophoresis through agarose. A Southern blot is prepared by transferring the size-separated DNA fragments from the gel to a nylon or nitrocellulose sheet. A sequence of interest can be detected on the blot by hybridization with an appropriate homologous radiolabeled “probe” sequence, followed by autoradiography. A dark band will appear on X-ray film exposed to the blot at each site where the probe hybridized to homologous sequences. This technique has been applied in several ways to study lineage and clonality. Genes which naturally undergo rearrangement during differentiation can be used as markers of specific cell types. For instance, during maturation of B lymphocytes the Ig genetic regions recombine to align one of each of several noncontiguous DNA segments to create a functional Ig gene for transcription (reviewed in Waldmann et al., 198513). In contrast, the characteristic band pattern reflective of the germ-line DNA configuration persists in nonlymphoid cells. Because the process of Ig gene rearrangement is normally limited to those cells committed to the B-lymphocyte pathway, DNA analysis can yield information regarding the cell type of the neoplasm. The finding of rearranged Ig genes in tumor tissue suggests the neoplasm is of B-cell origin.

3. T-cell Receptor Gene Loci A process similar to Ig gene rearrangements occurs during maturation of T-lymphoid cells when the T-cell receptor genetic regions recombine to form a functional gene for transcription (see Section 111,E).Thus, the finding of rearrangement in the T-cell receptor locus

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might indicate a tumor of T-cell lineage. Normal lymphoid tissues contain cells with myriad individual Ig or T-cell receptor rearrangements. The finding of many cells with the same DNA rearrangement in a neoplasm suggests that it developed clonally. B. X-CHROMOSOME INACTIVATION MOSAICISM

1 . Glucose-6-phosphate Dehydrogenase (G6PD) A very useful method for tracing the development of human neoplasms takes advantage of X-chromosome inactivation mosaicism with GGPD as a cell marker. The technique was first used by Linder and Gartler (1965) in a study of uterine leiomyomas. These workers examined tumors arising in women who were heterozygous for two forms of the enzyme GGPD. The GGPD locus is within a region of the X chromosome that undergoes inactivation in somatic cells with more than one X. This process occurs early in embryogenesis and is random and stably heritable. That is, the paternaIly and maternally derived X chromosomes are equally likely to be inactivated in a given cell and the same pattern of inactivation characterizes all the progeny of that cell. Women who are heterozygous at the GGPD locus (Gd) for the common B allele (GdB) and a variant such as GdAhave two populations of cells, one in which GdBis active and B-type enzyme is produced and the other in which GdA is active and GGPD A is synthesized. In a GdBIGdAwoman, a tumor that arises from a single cell exhibits a single-enzyme phenotype, whereas one that arises from many cells is very likely to show both GGPD types. Although the vast majority of Caucasians carry only the B allele, about 35% of black women are heterozygous for B and either the A or A- variant. The B and A enzymes are readily distinguishable because they have different electrophoretic mobilities, whereas the A- and A enzymes have similar or identical patterns of migration. The A- enzyme is more rapidly degraded than B or A, and only low activity levels are present in aging anucleate red blood cells. However, near normal enzyme activity levels are maintained in nucleated cells. The sensitivity of the assay system is such that as little as 5% of the minor component in a mixture of B and A can be detected (Fialkow, 1973). Furthermore, the GGPD phenotype of as few as 30 granulocytes and erythroblasts, 4-6 megakaryocytes, 2-3 macrophages, or 50-100 T cells can be defined (Lim et al., 1984). In order to interpret fully the GGPD phenotype of a tumor, and ratio

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of B to A enzyme in the normal tissue from which it arose should be known. In a study of more than 100 G6PD heterozygotes with nonleukemic neoplasms, a close correlation in the ratios of GGPD A to B was noted in multiple mesenchymal cell types from the same individual (Fialkow, 1983, and unpublished observation). Several possibilities must be considered when interpreting the G6PD phenotype of a tissue (see Fialkow, 1972, for detailed discussion):

1. The observation of a double-enzyme phenotype might indicate that the sample is not neoplastic. However, the presence of a significant admixture of normal tissue components could obscure a clonal neoplastic population. Many solid tumors contain normal elements such as stroma, blood cells, and normal parenchymal cells; lymph nodes may contain normal lymphocytes as well as lymphoma cells. Because certain cell types contain more G6PD activity than others, even minor admixture of normal cells of high activity with clonal neoplastic cells of different lineage might result in a double-enzyme phenotype and wrongly suggest polyclonal development of the neoplastic process. Therefore, cellular homogeneity of the assay sample is of great importance. Alternatively, a neoplasm with a double-enzyme phenotype could have arisen from transformation of many cells, that is, it could be a polyclonal tumor. 2. The observation of a single-enzyme phenotype must also be interpreted with caution. Although it could signify a neoplasm that developed from a single cell, the possibility exists that transformation occurred in several cells, all bearing the same active X chromosome. Clearly, the more cells involved in the initiation of the neoplasm, the less likely is a single-enzyme phenotype to be found. Another possibility to explain a single-enzyme phenotype is postinactivation selection. This type of selection occurs in women who are heterozygous for a gene that when present in boys causes the X-linked recessive Lesch-Nyhan syndrome. These women have the normally active allele for hypoxanthine phosphoribosyl transferase (HPRT) on one X chromosome and an allele that causes severe deficiency of HPRT activity on the other X. Whereas tissues such as skin show the expected intermediate enzyme activity between normal and mutant levels, hemopoietic cells manifest normal activity levels (Dancis et al., 1968; Nyhan et al., 1970). This occurs because blood cells with the mutant HPRT allele active suffer strong selective disadvantage. Mutant alleles for other X-linked genes may also result in strong negative selection (Prchal et al., 1980; Gealy et al., 1980; Migeon et al., 1981; Conley et al., 1986). In a woman doubly heterozygous for such a locus

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and GGPD, the finding of a single enzyme-phenotype would not necessarily imply the presence of a clonal neoplasm. These situations are found infrequently. For example, only 2 of 241 G6PD heterozygotes without leukemia studied in our laboratory who exhibited a doubleenzyme phenotype in skin showed a single G6PD type in blood granulocytes (Fialkow, 1973; Fialkow et d . ,unpublished). 3. Since G6PD is a dimeric protein, composed of two molecules, reactivation of the inactive X chromosome in a heterozygote would result in production of a heteropolymer, with migration on the gel midway between the A and B positions (Yoshida et al., 1967; Silagi et al., 1969). Aside from oocytes, activity of both X chromosomes in a given cell has been observed only in abnormal situations, such as interspecific hybrids subjected to selective pressure (reviewed in Gartler and Riggs, 1983). In addition to direct assays of cell populations, hemopoietic diseases may be studied by analyzing colonies that develop when blood or marrow cells are cultured in semisolid media. For proper interpretation of the data it is important to know whether these colonies are true clones. In 1976 it was shown with G6PD that human erythroid colonies grown in vitro from normal heterozygous women arise from single cells following plating at low density (Prchal et al., 1976). This finding was later extended to granulocyte/macrophage, T-cell, and mixed-cell colonies (Singer et al., 1979a, 1981; Powell et al., 1984). Clonally derived mixed-cell colonies containing lymphocytes as well as myeloid elements have been reported (Lim et al., 1984). This finding is consistent with the existence of a common progenitor for lymphoid and myeloid cells (see below). As plating concentration increased, however, so did the proportion of colonies showing both A and B enzymes (Singer et al., 1979a; Powell et al., 1984). Therefore, quantitation must be carefully interpreted to avoid underestimation of the number of colony-forming units and concomitant overestimation of the proportion of mixed-cell progenitors (CFU-GEMM).

2 . Restriction Fragment Length Polymorphisms (RFLPs) DNA in intergenic noncoding regions can also serve as a marker of a clone. Since restriction endonucleases recognize specific DNA sequences, single base variations in DNA can create or abrogate an endonuclease recognition site. The latter change alters the band pattern that results when DNA is digested with that endonuclease and run on a gel that separates molecules by size. Naturally occurring polymorphisms for these base substitutions are widespread thsough-

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out the genome. The resultant restriction fragment length differences are detectable with appropriate probes (Bostein et al., 1980). In and of themselves, RFLPs are not useful for determining clonality since each cell in a heterozygote has both DNA sequences. However, just as GGPD can be exploited to trace lineage and clonality by enzyme patterns, X-linked RFLPs which affect methylation patterns yield similar information by Southern blot patterns (Vogelstein et al., 1985). Homologous DNA regions on the active and inactive X chromosomes often differ by degree of methylation of cytosine residues. Certain restriction endonucleases are methylation sensitive. That is, some will only recognize a site if the cytosines are methylated, whereas others require unmethylated cytosines for recognition. Heterozygosity for an X-linked RFLP allows identification of the maternally and paternally derived X chromosomes by digestion with one restriction endonuclease, and digestion with a methylation-sensitive endonuclease enables the active and inactive Xs to be distinguished if there are appropriate methylation differences. The major advantage of this system is that potentially tumors from virtually every female are informative. It is estimated that 27% of women are heterozygous at the HPRT locus for one particular RFLP identifiable by digestion with the endonuclease known as BamHI (Yen et al., 1984; Wolf et al., 1984). The use of additional X-linked probes will increase the likelihood that a given patient will be found to be heterozygous for at least one X-linked RFLP. C. CYTOCENETICS The occurrence of nonrandom cytogenetic abnormalities in neoplastic cells, first recognized in CML (Nowell and Hungerford, 1960), is now known to be common. As better banding techniques were devised, specific chromosome markers have been associated with a variety of neoplasms, “solid” as well as hematologic. The finding that all the neoplastic cells isolated from a patient share a common abnormal karyotype suggests that the tumor developed clonally. It is theoretically possible, however, that an agent involved in causation of the disease might have specific affinity for certain chromosomal regions, in which case finding the same karyotypic change in each cell would not necessarily indicate clonal development. In neoplasms that are cytogenetically heterogeneous, the presence of structurally rearranged marker chromosomes supports divergence from a common stemline as a result of the karyotypic instability inher-

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ent in many malignancies. Therefore, the presence of a chromosomal abnormality does not prove that the cytogenetic alteration was the initial, or even an essential, event in development of a disease, but it does provide a useful marker for following the clone it identifies. Less common but informative chromosomal mosaicism occurs constitutionally. An individual may have karyotypically distinct cell populations, usually due to a nondisjunctional event early in embryogenesis, for instance, 47,XXY/46,XYor 46,XX/45,Xmosaicism. In such an individual, the finding that all tumor cells are derived from only one of the two constitutional lines suggests that the neoplasm developed from one or a few cells. Addition or loss of a chromosome or segment can be detected in some cases by changes in intensity of a band on a Southern blot or disappearance of the band corresponding to one allele (Cavenee et al., 1983; Fearon et al., 1986). Ill. Lymphoproliferative Disorders

B lymphocytes develop by an ordered progression of differentiation from pluripotent stem cell to Ig-secreting plasma cell (reviewed in Calvert et al., 1984).The earliest identifiable cells belonging to the B lymphocyte lineage are called pre-B cells. These cells have rearranged DNA coding for the Ig heavy chain but do not produce Ig heavy or light chains. They give rise to other pre-B cells that contain cytoplasmic but not surface-linked heavy chains and may also bear Blymphocyte-associated non-Ig surface markers. The K and A light chain genes then undergo rearrangement in a hierarchal scheme, such that a single Ig light chain class from only one of the homologous chromosomes is expressed. The cell is termed a B lymphocyte when Ig is incorporated into its plasma membrane. The first class of Ig to be expressed on the developing B cell is IgM. Although the cell may eventually switch expression to another isotype, the heavy chain variable region and the light chain remain the same. When a mature B cell is activated by exposure to a suitable antigen it begins to proliferate and to secrete Ig. Some of the progeny of these activated B cells become terminally differentiated plasma cells with limited lifespan, whereas others revert to small long-lived lymphocytes, the so-called memory cells.

A. MULTIPLEMYELOMA The different B-lymphoid neoplasms are characterized by predominance of one or another of these cell types. For example, in multiple

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myeloma there is an increase in the percentage of antibody-secreting plasma cells. Although there are billions of plasma cells, the secreted Ig molecules have the same light chain and idiotype, consistent with the hypothesis that the functional plasma cells develop clonally (e.g., Mellstedt et al., 1974). Studies of three patients with myeloma and one with a plasmacytoma who were heterozygous for GGPD confirmed the clonality of these neoplasms (reviewed in Fialkow, 1974). A population of circulating B lymphocytes bear on their surfaces the same Ig as is found free in the blood (Mellstedt et al., 1974; Abdou and Abdou, 1975; Kubagawa et al., 1979). These findings indicate that multiple myeloma is a disease of clonally proliferating B lymphocytes able to differentiate into Ig-secreting plasma cells.

B. B-CELLCHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND NON-HODGKIN’S LYMPHOMAS (NHL) Many lines of evidence support the conclusion that B-cell CLL and NHL are clonal disorders of progenitor cells with differentiative expression restricted to the B-lymphoid pathway. In marked contrast to normal populations of blood and lymph node lymphocytes in which there are mixtures of K and A light chain-producing cells (usually in a 2 to 1ratio), a single light chain type is produced by the vast numbers of malignant cells in the B-cell chronic neoplasms (Johansson and Klein, 1970; Aisenberg and Bloch, 1972). The heavy chain class and idiotype are similarly monotypic in CLL, whereas there is extensive diversity in the antibody idiotypes detectable in reactive proliferations of B lymphocytes (Froland and Natvig, 1972; Salsano et al., 1974; Schroer et al., 1974). Analyses of hemopoietic compartments in GGPD heterozygotes with CLL confirm the clonality of the neoplastic B lymphocytes, which express only a single GGPD allele. In contrast, both alleles are expressed in proportions similar to skin in granulocytes, erythrocytes, platelets, and at least some T lymphocytes, indicating that they do not differentiate from the leukemic progenitor cells (Fialkow et al., 1978a). Unlike multiple myeloma where plasma cells differentiate from the neopIastic done, in CLL there is restriction in the maturation of the clone to the mature B lymphocyte stage. Using flow cytometric techniques, Smith et al. (1984) and Berliner et al. (1986a) were able to detect small populations of circulating lymphocytic cells belonging to the neoplastic clone in SO-80% of patients with B-cell Iymphomas, even early in the course of the disease. The clonal cells perturb the shape of the histogram that describes the distribution of fluorescence staining for surface K and A

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light chains and may be useful in the diagnosis and surveillance of lymphoid malignancy. Ig gene rearrangements represent early specific events in B-cell differentiation and serve as DNA markers for commitment to the Bcell lineage prior to production of antibodies and other cell surface lineage determinants. In the normal situation, hybridization of an Ig probe to electrophoresed DNA from a population of lymphocytes results in a diffuse smear on the Southern blot since no single Ig DNA configuration is present in great enough quantity to produce a discrete band. A specific gene rearrangement, the molecular equivalent to a serologic idiotype, will only be detectable by Southern blot analysis when a clonal subpopulation is present (Korsmeyer et al., 1982; Arnold et al., 1983). The DNA method offers a significant advantage over serologic techniques since a minority population of clonal cells comprising only 510%of the sample is sufficient to produce a band on the Southern blot. Arnold et al. (1983) were able to identify monoclonal B-cell populations within lymphomas bearing ambiguous surface markers or containing admixtures of reactive T cells. In a patient in whom the tumor cells were morphologically too undifferentiated to allow distinction between lymphoma and carcinoma, B-cell lymphoma was diagnosed on the basis of the presence of a clonal Ig gene rearrangement, allowing appropriate therapy to be planned. Instances have been reported in which two distinct idiotypes have been found in a single patient (Sklar et al., 1984; Kitchingman et al., 1984; van Dongen et al., 1984; Giardina et al., 1985). One explanation for these findings is that these tumors are truly “biclonal,” that is, two cells gave rise to the tumor by independent transformation events. It seems more likely, however, that the tumor is monoclonal, arising in an immature lymphoid progenitor in which the Ig loci continue to undergo rearrangements; as differentiation proceeds within this clone, two or more subclones with different Ig DNA rearrangements develop (Fialkow, 1985).Another possibility is alteration of the initial idiotype by somatic mutation or posttranscriptional modification. The occurrence of more than one idiotype in cases of B-cell neoplasia has implications for therapy. For example, a specific cytolytic monoclonal antibody raised against the Ig idiotype expressed by the major population of tumor cells might not be effective against neoplastic cells with other idiotypes in the same tumor. In fact, in 1982, successful use of a cytolytic anti-tumor-cell-idiotype monoclonal antibody was described (Miller et al., 1982); subsequently, however, failures to achieve lasting remission were often seen (Raffeld et al., 1985;

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Meeker et al., 1985). Detailed analysis of the Ig genes in some patients whose tumor cells underwent immunologic change revealed no new rearrangements in the Ig DNA, strongly suggesting that loss of anti-idiotype reactivity resulted from changes, such as base substitutions, not detectable by tests using available restriction enzymes (Raffeld et al., 1985). These results are consistent with existence of a common progenitor for the lymphoma cells at both stages of disease and development of new immunologic reactivity by emergence of a subclone. Further evidence supporting the occurrence of clonal divergence comes from study of a patient with CML whose blast cells during two different lymphoid crises displayed different arrangements in light chain Ig DNA, yet contained identical heavy chain gene rearrangements. Additionally, cytogenetic analysis revealed monosomy 7 during both blast phases, but not in the intervening chronic phase (Bakhshi et al., 1983). Other evidence supporting the subclonal evolution hypothesis was found in acute lymphocytic leukemia (Kitchingman et al., 1986; see Section 111,D). The strongest evidence for clonal evolution was obtained by examination of serial biopsies from B-cell follicular lymphoma patients whose tumors were characterized by t(14;18) (Raffeld et al., 1986).In the three cases in which Ig gene patterns changed but the t(14;18) breakpoint remained unaltered, the variation could always be accounted for by secondary rearrangements, indicating that the various neoplastic subpopulations arose from a common progenitor cell. In the event that two completely unrelated Ig gene rearrangements are found, proof of biclonality could be obtained by using a second marker system, such as X-chromosome inactivation mosaicism with G6PD or an X-linked RFLP as a marker. The finding that the tumor contains a mixture of cells with the paternal and maternal X chromosomes active would document the coexistence of independent clones. C. OTHERCHRONICB-CELLLYMPHOPROLIFERATIVE DISORDERS Waldenstrom macroglobulinemia is a disease similar to multiple myeloma. In the former neoplasm monoclonal IgM is produced (Preud’Homme and Seligmann, 1972), whereas in the latter the antibody class is usually IgG or occasionally IgA. In heavy chain disease, fragments of monoclonal Ig heavy chains are secreted (Seligmann et al., 1968). Immunoblastic lymphomas that develop in patients with one of these chronic B-cell disorders bear surface Igs identical to those secreted during the chronic phases, suggesting that the lympho-

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mas evolve from the original heavy chain disease neoplastic clones (Brouet et al., 1976, 1977; Ramot et al., 1977). Until recombinant DNA techniques became available, hairy cell leukemia cells defied categorization, having properties in common with B cells, T cells, and monocytes (Golomb et at., 1978; Jansen et al., 1982; Scheinberg et al., 1978). The presence of both heavy chain and light chain gene clonal rearrangements shows that many if not all hairy cell leukemias are clonal B-cell diseases (Korsmeyer et al., 1983a). Clonal development of Burkitt lymphoma has been demonstrated using a variety of cell markers including Ig and GGPD (Fialkow et al., 1973; reviewed in Fialkow, 1980) and chromosomal translocations and oncogene rearrangements (reviewed in Leder et al., 1983; Waldmann et al., 1985a). Burkitt lymphoma responds well to remissioninducing chemotherapy. However, relapses occur in more than half of the patients. GGPD and Ig phenotyping show that early relapses result from reemergence of the originally detected tumor clone, whereas some late recurrences, although clonal in nature, are independent malignancies (Fialkow et al., 1972, 1973). Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Burkitt lymphoma (reviewed in Epstein and Achong, 1979),although the role of the virus in inducing this malignancy is not well understood. Perhaps through EBV transformation a large pool of preneoplastic B cells is immortalized, providing a population of cells susceptible to the additional changes that result in Burkitt lymphoma. More than one independent malignancy might then be expected to occur in some individuals. Although this malignant disease develops clonally, benign virally induced growths, such as condyloma accuminata, have been shown to be multicellular in origin (Friedman and Fialkow, 1976). EBV has also been associated with neoplastic lymphoid proliferations in immunosuppressed patients following marrow or organ transplants (e.g., Frizzara et al., 1981; Reece et al., 1981; Cleary et al., 1984; Cleary and Sklar, 1984), in patients with inherited immunodeficiencies (e.g., Shirley et al., 1982), and in patients with acquired immunodeficiency syndrome (Groopman et al., 1986). These proliferations are often multicentric, and most individual tumors are monoclonal although the tumors at different sites are independent. However, in some cases, single tumors have been suggested to be oligoclonal or polyclonal by Ig protein or DNA analyses (Hanto et al., 1981; Shearer et al., 1985; Cleary and Sklar, 1984), especially when the malignancies were studied soon after onset of illness. Although it is possible that the nonhomogeneous phenotype resulted from sub-

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clonal evolution of a single clone, the observation that some tumors studied sequentially become monoclonal suggests that these EBVmediated lymphomas begin as transformation of many cells, with subsequent evolution to monoclonality, reminiscent of the pattern seen in EBV transformation of B lymphocytes in uitro (Behcet et al., 1974).

D. ACUTE LYMPHOCYTIC LEUKEMIA (ALL) Several classifications have been devised for ALL. One system is based on the surface phenotype of the leukemic cells-the presence of antigens, antibodies, or receptors consigning the cells to T- or Bcell lineage. Leukemias comprised of cells lacking distinguishing surface markers are called “null-cell” leukemias. The term “common ALL” is sometimes used when referring to leukemias in which the cells bear on their surfaces the common ALL antigen (CALLA). A small minority of ALL cases fall into the B-cell category when judged by these surface characteristics. However, many cases of common and null-cell ALL in fact might be of B-cell lineage since molecular analyses reveal Ig gene rearrangements in the leukemic cells (e.g., Korsmeyer et al., 198313; Foa et al., 1984). Using surface antigens to distinguish T- and B-cell ALL, Korsmeyer et al. (1983b) found that all 25 cases lacking definite T-cell surface markers contained Ig gene rearrangements, whereas all 12 cases with T-cell markers retained Ig genes in the germ-line configuration. Therefore, the non-T leukemias appear to be proliferations of cells blocked in early stages of B lymphocyte differentiation, before onset of manufacture of Igs. Recently, however, it has been found that the same malignant cell may display characteristics of more than one lineage and, therefore, that precise definition of lineage may not be possible in some of these lymphoid leukemias. Among the explanations for the ambiguous cases are the following:

1. Accurate categorization of a cell population is limited by the sensitivity and specificity of the marker used. For instance, the monoclonal antibodies used to distinguish T- from B-cell neoplasms may not be as specific markers as was once thought; although some antigens are primarily found on T cells and others primarily on B cells, there is some overlap. Similarly, Ig heavy chain gene rearrangements have been occasionally found in cells other than B-lymphoid cells and may be less specific than light chain DNA rearrangements as markers for the B-cell lineage (e.g., Forster et al., 1980; Kemp et al., 1980; Cory et al., 1980).

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2. It has been suggested that there is a transient stage in hemopoietic stem cell differentiation during which limited promiscuity of gene expression occurs (Greaves et d., 1986). Leukemic transformation of a cell at this stage of development, prior to irreversible lineage determination, might result in leukemia of ambiguous phenotype. 3. Changes in some malignant cells may alter lineage-specific differentiation programs, resulting in cells that express characteristics of more than one lineage (“lineage infidelity”) (McCulloch, 1983), thereby further confusing the classifications. One or a combination of these possibilities might explain the observation by Tawa et al. (1985) that of the 39 patients with null-cell leukemia who had clonal rearrangements in the Ig heavy chain genes, approximately 25% also evidenced rearrangements in the newly identified T-cell receptor DNA regions. Lineage ambiguity has also been observed in approximately 10% of cases classified as B-cell neoplasia by presence of cytoplasmic Ig heavy chains or B-cell-associated surface markers (Waldmann et al., 1985a; O’Connor et al., 1985; Pelicci et al., 1985). Dow et al. (1985)studied 19 patients with ALL who were heterozygous for G6PD; 6 patients had common-type ALL, 2 had pre-B-cell, 4 had undifferentiated, 4 had T-cell, and 3 had undefined ALL, categorized on the basis of surface markers. Seven patients were evaluated only at diagnosis, nine only at relapse and three both at diagnosis and at relapse. During active disease, lymphoblasts from each patient exhibited a single G6PD phenotype, whereas nonlymphoid blood or marrow components revealed the same G6PD mosaicism as was seen in skin. Ferraris et al. (1985a) obtained comparable findings in 3 patients heterozygous for the Mediterranean variant of G6PD ( GdBl GdMed)studied at initial presentation with ALL. These data suggest that the ALLs are clonal diseases and that differentiative expression of the leukemic progenitors is apparently restricted to the lymphoid pathway. Kitchingman et aZ. (1986) reported 18 ALL patients in whom more than two discrete heavy chain Ig bands were detected on Southern blots. In seven cases, the multiplicity of heavy chain bands correlated with extra copies of chromosome 14 (which contains the heavy chain loci). Thus, the leukemia most likely arose in a cell which had not yet rearranged its heavy chain Ig genes but had gained additional chromosomes 14. Leukemic descendants were capable of undergoing heavy chain gene rearrangements on each chromosome 14, even if more than two of this chromosome were present. Although the other 11cases might represent occurrences of biclonality, the presence of a

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single stem line karyotype suggested a clonal leukemia with subclonal evolution. In the patients studied by Dow et al., samples were tested during two separate periods of active disease in 6 cases (3 at diagnosis and relapse and 3 during two relapse phases). In each instance the monomorphic expression of GGPD in lymphoblasts obtained during both active phases was the same. During remission mononuclear hemopoiesis was nonclonal in the 17 patients tested. The latter observation indicates that during remission the marrow is IargeIy populated by normal cells (see Section IV,C,3). However, the finding that the same clonal disease recurs at subsequent relapses indicates persistence of small numbers of leukemic cells during remission in some patients. In 3 or 7 patients studied by Zehnbauer et al. (1986) residual leukemic cells were detected during clinical remission using Ig gene rearrangements as clonal markers. The method used was sensitive enough to detect 1 leukemic cell per 500 nucleated marrow cells and should prove a valuable tool in evaluating the level of remission achieved.

E. T-CELLLYMPHOPROLIFERATIVE DISORDERS Just as Ig gene rearrangements have been used as markers of B-cell lineage and clonality, receptor gene rearrangements have proved useful in the study of T-cell neoplasia. The situation is analagous to the Ig system with its heavy and light chain and constant and variable region components. The T-cell receptor is a surface structure that allows the T cell to recognize an antibody-bound antigen. This receptor, composed of three chains denoted a,p, and y, is formed during T-lymphocyte differentiation (e.g., Saito et al., 1984; Ohashi et al., 1985; reviewed in Acuto and Reinherz, 1985). Diversity of T-cell antigen recognition ability is governed in part by complex somatic alterations in the noncontiguous DNA segments that code for the a and p chains (T, and Tp) (Saito et al., 1984). As a consequence of T-cell receptor DNA processing, a portion of the Tp constant region is deleted. DNA from cells of the T-lymphocyte lineage have a deleted or markedly diminished band that can be detected with a Tp constant region probe, whereas non-T lymphocytes retain the germ-line pattern (Flug et a1., 1985; Waldmann et al., 1985a; Tawa et al., 1985).Therefore, absence or diminution of this constant region Southern blot band is a marker for cells of T-lymphocyte lineage. The polyclonal Tp variable region rearrangements in normal T-cell populations are not detectable as discrete bands on Southern blots since the individual changes are too numerous and each is present in a concentration that is below the

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limit of resolution of the method. Only when a clonal population of T lymphocytes is present will a detectable band form. A variety of T-cell neoplasms has been studied with this technique. Rearrangement of the Tp gene was apparent in almost all cases otherwise classified as T-cell malignancy. For example, T, gene rearrangements were detected as discrete new bands on Southern blots prepared from 26 of 27 tumor samples considered to be of T-cell derivation by surface marker analysis and also from 3 established Tcell ALL-derived lines (Flug et al., 1985; Waldmann et al., 198513). However, 3 of 19 T-cell lymphomas reported by O’Connor et al. (1985) retained the germ-line Tp chain Southern band pattern. It is possible that these cases represent polyclonal neoplasia with no one subclone present in great enough proportion to produce an identifiable band. Other possibilities are that the rearranged T-cell receptor gene in the malignant clone has been deleted or that only the T, or T, chains have rearranged. It is also possible that the stem cell responsible for the proliferation may be unable to undergo the receptor gene rearrangement, yet causes disease that resembles T-cell neoplasia. In any event, with some exceptions, deletion or diminution of the Tp constant region restriction fragment is a marker for T-cell lineage, and detection of specific rearrangements is evidence for clonal expansion. This system is particularly informative when the neoplastic cells are very immature and identification by histochemical or immunological methods is difficult. However, there is considerable heterogeneity within the lymphocytic leukemias classified in this manner-T-lineage cells with only T-cell receptor gene rearrangements, T-lineage cells with both T-cell receptor and Ig gene rearrangements, B-lineage cells with only Ig gene rearrangements, and B-lineage cells with both Ig gene and T-cell receptor gene rearrangements. In addition, there are some leukemias in which the lineage of the proliferating cells cannot be determined with certainty using techniques currently described. Lymphocytes bearing a surface antigen called T8 function as suppressor-killer cells (Reinherz et al., 1980).Recently, a syndrome characterized by neutropenia, anemia, and lymphocytosis of large granular T8-positive lymphocytes has been described (Reynolds and Foon, 1984). The underlying defect in this disease is not known. If the lymphocytosis were polyclonal, it could have resulted fr0m.a nonneoplastic immune response, whereas clonal lymphocytosis would be more compatible with neoplastic expansion. Several groups examined the TBgenes in lymphocytes from patients with this syndrome and found clonal patterns of rearrangement, strongly suggesting that prolifera-

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tion of a single clone of T8 cells is important in the pathogenesis of the disease (Waldmann et al., 1985b; Rambaldi et al., 1985; Aisenberg et al., 1985; Berliner et al., 1986b). However, in some cases of T8 lymphocytosis, monoclonality could not be proved, and this category of disease may also be more heterogeneous than was initially thought (O’Connor et al., 1985; Foa et al., 1986). Waldmann et al. (1985b) used the Tp clonal rearrangement as a marker for the malignant clone to study the patterns of remission and relapse in a patient with T-cell ALL. The patient’s malignant cells reacted with anti-Tac antibody, an antibody that recognizes the interleukin 2 receptor (Leonard et al., 1982); immunotherapy with anti-Tac antibody successfully eliminated Tac-positive cells from the circulation. To distinguish between true remission and mere loss of the Tac reactivity, sequential molecular studies of the patient’s peripheral blood lymphocyte Tp region were performed. Southern blot analysis of cells obtained during first relapse revealed a rearrangement in the Tp gene. The Southern blot band disappeared following therapy, a finding consistent with absence of clonal lymphopoiesis and existence of a true remission. Mycosis fungoides is a T-cell malignancy with predilection for involvement of the skin and lymph nodes. Diagnosis may be very difficult, with neoplastic alterations masked by chronic inflammatory changes. Evaluation of T-cell receptor gene rearrangements has proved to be a practical method for detecting mycosis fungoides cells in biopsy specimens. Weiss and co-workers (1985) studied lymph node samples from 14 patients with that disease. Clonal rearrangements were detected in all 8 lymph node specimens containing histologically unambiguous mycosis fungoides cells. An additional 9 lymph node specimens were described as “benign lymphadenopathy” by routine pathologic study. However, clonal T-cell receptor rearrangements were found in 7 of these 9 specimens. There is a spectrum of T-cell lymphoproliferative syndromes associated with the human T-cell leukemia virus (HTLV) (reviewed in Broder et al., 1984). This RNA virus directs the production of a DNA copy which integrates, probably at random, in the genome of the infected cell. The cellular DNA sequences attached on either end of the integrated virus contain restriction endonuclease sites at varying distances from the viral genome they border. Therefore, the size of the DNA fragment containing the viral sequences will differ for different integration sites. Analyses of the Southern blot band patterns that result when restriction endonuclease-digested tumor DNA is probed with a sequence specific for the viral genome reveals that the tumors

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are monoclonal or oligoclonal, since only one or a few discrete bands are found. These very aggressive virally induced diseases may initially be polyclonal with subsequent overgrowth by a single dominant clone.

IV. Myeloproliferative Disorders

A. CHRONIC MYELOCYTICLEUKEMIA (CML)

1 . Clonal Development CML is a chronic myeloproliferative disorder characterized by marked overproduction of myeloid cells. In the chronic phase of the disease, functional differentiated cells of all hemopoietic lineages are produced. When the disease enters the accelerated or blast phase, maturation of marrow cells is deficient and blasts accumulate; although aggressive chemotherapy may prolong life, the patient invariably succumbs. CML was the first human malignancy for which a specific chromosome abnormality was identified (Nowell and Hungerford, 1960). This characteristic marker, the Philadelphia chromosome (Ph), results in most cases from a reciprocal translocation between chromosomes 9 and 22 (Rowley, 1973). Approximately 90% of patients with clinical CML manifest Ph in their leukemic cells. Differences in duration of survival and response to therapy suggest that some of the 10% of patients with Ph-negative CML have a disease different from the Phpositive leukemia. The observation that Ph is present in 90-100% of marrow metaphase cells from untreated chronic phase Ph-positive CML patients suggested the clonal nature of the disease. Strong evidence in support of this hypothesis was provided by GGPD studies. Thirty-two GGPD heterozygotes exhibited a double-enzyme GGPD phenotype in skin and cultured skin fibroblasts, but only a single enzyme type was found in blood granulocytes (14 patients had type A and 18 patients had type B) (Fialkow et al., l967,1977,1978b, unpublished; Fialkow, 1985).Although there are situations in which there is selection against hemopoietic cells whose active X bears a deleterious gene (Section II,B,l), such instances are uncommon and could not have accounted for the skewing seen in all these CML cases (or the skewing seen in other myeloid proliferations discussed below). The conclusion based on GGPD studies that CML develops clonally is supported by cytogenetic observations. For example, studies

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of patients in whom the parental origins of each member of a chromosome number 22 (or 9) pair were distinguishable by banding polymorphisms showed involvement of the same chromosome in the translocation in every informative cell, arguing against generation of Ph in many cells by multiple translocation events (Gahrton et al., 1974; Hayata et al., 1974; Hossfeld, 1975). In approximately 10% of cases, CML leukemic cells are marked by a variant translocation, involving chromosomes different from or additional to 9 and 22. Occasionally these changes result in a “masked” Ph in which the classic Ph morphology is not recognizable (reviewed in Sandberg et al., 1985). In these cases, the presence of the unusual karyotypic change in the majority of unstimulated blood or marrow metaphase cells also supports a clonal origin of the translocation. CML patients with constitutional sex chromosome mosaicism have been reported (Tough et al., 1961; Fitzgerald et al., 1971; Chaganti et al., 1982). If the event creating Ph occurred more than once it might b e predicted that both stem lines would be affected. However, Ph was associated with only one of the stem lines in each case. Other evidence for clonality of CML was obtained with alleles of 6phosphogluconate dehydrogenase as cell markers (Fialkow et al., 1969) and with a somatic cell hybridization technique (Guerts van Kessel et aE., 1982). I n the latter study leukemic cells from a patient with CML who was heterozygous for an enzyme polymorphism mapped to chromosome 9 were fused to rodent cells. Hybrids retaining the 9q+ chromosome expressed one allele, whereas hybrids retaining the normal 9 chromosome expressed the other. It has been found that the sites of breakage on chromosomes 9 and 22 in the formation of Ph vary between patients (Groffen et al., 1984a; Leibowitz et al., 1985). However, all Ph-positive cells in a given patient have the same specific breakpoint within the “breakpoint cluster region” (bcr)on chromosome 22, consistent with clonal derivation of Ph.

2 . Dwerentiatiue Expression of the Stem Cell lnvolved by CML Ph has been found in erythrocytic marrow cells (Clein and Flemans, 1966; Rastrick et al., 1968). This observation and the finding of a single-enzyme G6PD phenotype in erythrocytes as well as in granulocytes indicate that CML involves a multipotent stem cell (Fialkow et al., 1967, 1977). The finding that platelets and monocytes from GdBI GdAheterozygotes with CML exhibit a single-enzyme type indicates that the involved marrow stem cell is multipotent for these lineages as we11 (Fialkow et al., 1977). Study of marrow cells cultured in semi-

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solid media provides other evidence supporting the involvement of multipotent stem cells by CML. With GGPD as a marker, it has been shown that under appropriate conditions these colonies arise clonally, from a single colony-forming cell. Monocyte/macrophage, red cell, eosinophil, and mixed-cell colonies from G6PD heterozygotes contained the same G6PD type found in granulocytes and granulocyte colonies, confirming that the CML stem cell can give rise to these hemopoietic elements (Singer et al., 1979b; Douer et al., 1983; Koeffler et ul., 1980). Cultured marrow fibroblasts from CML patients do not arise from the CML clone as they contain both GGPD enzyme types in the same proportion as skin. Compatible with the noninvolvement of marrow fibroblasts in chronic and accelerated phase CML is the finding that the myelofibrosis occurring in CML is not part of the clonal proliferation (Fialkow et al., 1977). Similarly, many investigators have reported that cultured marrow fibroblasts from patients with Ph-positive CML are Ph-negative (Maniatis et al., 1969; de la Chapelle et al., 1973; Fialkow et al., 1977; Greenberg et al., 1978).Therefore, myelofibrosis is a secondary, reactive process and not a direct result of clonal neoplastic growth. Although mitogen-stimulated lymphocytes from patients with CML are generally Ph-negative, the occurrence of lymphoid blast crisis in approximately 30% of patients suggested that the CML stem cell could differentiate along the lymphoid pathway (Boggs, 1974; Shaw et al., 1975; Rosenthal et al., 1977; Janossy et al., 1978; Le Bien et al., 1979). Studies with GGPD provided stronger evidence for subpopulations of mature B and perhaps T lymphocytes that arise from the CML clone (Fialkow et al., 197813). The involvement of B-lymphoid cells was later documented unequivocally in studies in which GGPD, Ph, or both were used as markers (Martin et al., 1980; Bernheim et al., 1981). For example, with a combination of surface anti-IgM immunofluorescence staining and Q-banding of mononuclear cells stimulated with polyclonal B-cell mitogens, IgM-positive Ph-positive cells were found in cuItures from patients with CML (Bernheim et al., 1981). Study of Epstein-Barr virus (EBV)-transformed B-lymphoid cell lines provided incontrovertible evidence that the CML stem cell is able to differentiate to B-lymphoid cells. EBV is a polyclonal activator of B lymphocytes that can also transform mature B-lymphoid cells. The long-lived lymphoid lines that result from EBV transformation of nonneoplastic lymphocytes are initially polyclonal but become homogeneous with prolonged culture (Behcet et al., 1974). Ph-positive Blymphoid lines have been established from peripheral blood of CML

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patients (Martin et al., 1980; Nitta et al., 1985; Yamada et al., 1985; Raskind et al., unpublished observations). However, the number of Ph-positive lines from each patient is small and the possibility exists that the translocation could have occurred de no00 in a susceptible cell following EBV infection. Recovery of B-cell lines bearing the identical complex Ph translocation as was found in myeloid cells from two patients makes de no00 translocation an extremely unlikely explanation (Martin et al., 1980; Ohyashiki et al., 1984). Further evidence for clonal involvement of B lymphocytes in CML was obtained by characterizing the GGPD phenotype and karyotype of independently EBV-transformed B-lymphoid lines from four GGPD heterozygotes with CML. Lymphoid lines that were homogeneous with respect to GGPD phenotype when first tested were obtained by transforming aliquots of peripheral blood mononuclear cells in 0.2-ml microliter wells. All Ph-positive B-lymphoid lines expressed the same GGPD allele as the leukemic myeloid cells (Martin et al., 1982; Raskind et al., unpublished observations). Both K and h light chain-producing Ph-positive lines were recovered, indicating that Ig restriction is not fixed in stem cells of the CML clone and that at least during the chronic phase these cells may be responsive to the induction factors which normally regulate B-lymphoid differentiation (Martin et al., 1982). Therefore, the clonally expanding stem cell can give rise to B cells able to contribute to antibody diversity. During the lymphoid blast phase, by contrast, uniform Ig expression is found. Bakhshi et al. (1983)examined the status of the Ig gene loci in nine episodes of lymphoid crisis and found clonal Ig gene rearrangements in eight of these, although no surface or cytoplasmic Ig products could be found. Therefore, these episodes of lymphoid blast crisis seemed to involve clonal proliferation of B cells blocked at an earlier stage of differentiation. These observations indicate that Ig restriction occurs at some stage between the pluripotent stem cell involved by CML and the lymphoblast that is present in lymphoid blast crisis. The evidence for involvement of T lymphocytes in CML is not as strong as that supporting B lymphocyte involvement. As mentioned above, mitogen-stimulated lymphocytes from patients with CML generally are Ph-negative. However, T lymphocytes are long-lived, and most may have antedated emergence of the CML clone. Furthermore, the presence of Ph in a T lymphocyte might abrogate its mitogen responsiveness. Several lines of evidence suggest that the pluripotent stem cell involved by CML can differentiate along the T-lymphoid pathway:

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1. A subset of T cells obtained by velocity sedimenting blood from each of two GGPD heterozygotes whose diseases were poorly controlled shared the clonal GGPD phenotype of the myeloid cells (Fialkow et al., 1978b). 2. Rarely, blast crisis cells have T-lymphoid characteristics (Palutke et ul., 1982; Griffin et ul., 1983; Herrmann et ul., 1984; Jacobs and Greaves, 1984; Allouche et ul., 1985). 3. In one patient whose blast crisis cells had attributes of immature T cells, clonal rearrangement of the Tp gene was present in addition to rearrangement within the breakpoint cluster region (Chan et al., 1986), the molecular equivalent of the cytogenetic Ph translocation (reviewed in Groffen et al., 1984b). 4. Nogueira-Costa et al. (1985) grew T-cell colonies from various mononuclear cell fractions from an untreated CML patient. Cells grown from all the fractions were 99-100% E-rosette positive (a property of T lymphocytes) and five of eight metaphase cells from one subpopulation were Ph-positive. However, since only a few mitoses could be examined, it was not possible to exclude the possibility that they represented rare contaminating myeloid cells. 5. In one of six GGPD heterozyous patients studied by Najfeld et al. (1985), all 76 T-cell lines cultured in uitro manifested only the CGPD type produced by the leukemic myeloid cells. 6. Fauser et al. (1985)cultured mixed-cell colonies from a patient with Ph-positive CML. T lymphocytes were identified within these colonies by reaction with T-specific monoclonal antibodies and expanded by subcloning in microtiter wells in the presence of phytohemagglutinin and T-cell conditioiied medium. Each secondary colony was characterized for T-cell antigen expression as well as karyotype. Although only one or two metaphase cells were found in each of 13 of 39 secondary colonies, all were Ph-positive. The evidence is thus very strong that single stem cells in CML can produce both lymphoid and myeloid progeny, at least in uitro. Phpositive CML stem cells in vivo may be much more likely to generate cells of myeloid than of lymphoid lineage, accounting for the rarity of Ph-positive T lymphocytes in blood. In any event, the stem cell involved by CM L is pluripotent for myeloid, erythroid, megakaryocytic, B-lymphocytic, and probably T-lymphocytic elements

3. Multistep Pathogenesis Evidence for a multistep pathogenesis of CML is provided by studies of three GGPD heterozygotes that suggest that there are cells in

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patients with CML that originate from the CML clone yet are Phnegative (Fialkow et al., 1 9 8 1 ~Raskind ; et al., unpublished observations). If Ph-negative B cells were not involved by CML one would expect to find the same ratio of B-cell lines expressing each enzyme type as is found in skin (see Section 11,BJ). However, the ratios of GGPD B to A in EBV-transformed Ph-negative lymphoid lines recovered from these patients were markedly skewed; in each case there was an excess of lines sharing the GGPD type of the CML clone, suggesting that at least some B lymphocytes derive from the leukemic stem cell. Since these lines were Ph-negative, this observation suggests that there are stem cells that proliferate clonally despite the fact that they do not have Ph. Similar evidence derives from studies by Najfeld et al. (1985), referred to above, in which each of 76 T-cell lines cultured in vitro from one patient with CML showed the GGPD type produced by the leukemic myeloid cells. Despite this finding, Ph was not detected in any of these lines. In one patient chromosomal abnormalities were found in 8 of 33 Phnegative B-lymphoid lines that manifested the GGPD type characteristic of the leukemic clone but in 0 of 14 lines that exhibited the other GGPD type (Fialkow et al., 1981~).Since EBV-transformed lines established from normal donors generaIIy remain karyotypically normal during the first year of in vitro culture (Steel et al., 1977),the findings of so many abnormal lines, each of which had the GGPD type of the abnormal clone, suggests that the putative Ph-negative progenitor clone in CML is genetically unstable. One manifestation of this genetic instability might be subsequent development of Ph. The existence of a Ph-negative stage in Ph-positive CML might provide an explanation for one case in which Ph, present in chronic phase, was absent in blast crisis (Hagemeijer et al., 1979), for one case in which both Ph-positive and Ph-negative blast cells coexisted in blast phase (Appelbaum et al., 1983), and for six cases in which marrow cells were Ph-negative at initial presentation but subsequently became Ph-positive (Hayata et al., 1975; Tanzer, 1977; Lisker et al., 1980, 1985). Thus, in the pathogenesis of CML there may be at least one step initiating clonal proliferation of genetically unstable Ph-negative stem cells. Subsequently, the Ph translocation event occurs, conferring further selective advantage in uiuo, and additional steps result in disease progression. CML is a disease that has several discrete clinical stages. In a sense, the chronic phase of CML is a “preleukemic” stage in that there is maturation of all hemopoietic lineages despite the enlarged pool of myeloid progenitors. Subsequently, the disease progresses to an

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“acute” phase during which blast cells predominate and maturation of other cellular elements declines. During this phase CML closely resembles acute leukemia. That this progression occurs by subclonal evolution of chronic phase cells is supported by cytogenetic and molecular studies. In cases where variant Phs were found in chronic phases, the presence of the same atypical or complex Ph rearrangements in blast crisis cells strongly supports the concept that blast crisis develops from a preexisting clonally proliferating chronic phase cell. Molecular studies also support a subclonal development of blast crisis from chronic phase cells. Although the Southern blot band pattern detected by probes for the bcr region varied between patients, the identical pattern was seen in chronic and acute phase cells from each of five individuals (Bartram et al., 1986; Zalcberg et al., 1986). Furthermore, analysis of blast crisis cells containing two Phs from a sixth patient revealed an amplified bcr fragment in addition to the altered bcr Southern blot bands from chronic phase cells. This latter observation is consistent with the occurrence of later molecular changes in cells from the chronic phase clone, perhaps conferring selective advantage and leading to accelerated disease. In about 80% of cases new karyotypic alterations develop in the Phpositive cells, most frequently an additional Ph or chromosome 8 or development of an isochromosome for the long arm of 17 (Rowley, 1975). If cytogenetic surveillance is frequent and detailed, subclones present during chronic phase can be detected, and one of these subclones eventually prevails in the accelerated or blast phase (Lowenberg et al., 1985). Evidence that blast crisis often results from evolution of a subclone of chronic-phase cells derives from Ig gene studies. Lymphoid blast crisis cells are monoclonal by Ig gene tests whereas chronic-phase lymphocytes are not. Additional evidence that subclonal expansion may be etiologic in progression of disease to blast phase is the observation that blast cells from two separate lymphoid crises in a patient contained the same heavy chain Ig rearrangement but different light chain gene configurations (Bakhshi et al., 1983).The precursor cell for the blast crisis episodes must have undergone heavy chain DNA rearrangement but retained the germ-line configuration of its light chain genes. Subsequently, the light chain genes underwent independent rearrangements in different progeny cells, in some of which additional oncogenic changes occurred. Thus, in this patient, there was a chronic phase characterized by cells bearing germ-line Ig genes, subclonal expansion of cells with processed heavy chain genes, and then further clonal expansion of two different subclones.

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4 . Remissions and Persistence of Normal Progenitors Conventional chemotherapy generally does not alter the percentage of Ph-positive marrow cells, despite lowering the white blood cell count and increasing the percentage of normal appearing hemopoietic cells. Furthermore, single-enzyme phenotypes persist in G6PD heterozygotes following cytoreductive therapy (Fialkow et al., 1977; Singer et al., 197913). These observations indicate that true “remission” is not achieved, since the marrow is not repopulated by normal stem cells. Following aggressive chemotherapy, however, an increase in the proportion of Ph-negative marrow metaphase cells may be seen (Goto et al., 1982), and G6PD studies on one such patient showed that these Ph-negative cells can arise from nonclonal, presumably normal, stem cells (Singer et al., 1980). Furthermore, in some patients Ph-negative myeloid and erythroid colonies appear after prolonged in vitro culture, supporting the hypothesis that normal stem cells persist, perhaps in a suppressed state, in CML patients, even when cytogenetic evaluation of marrow reveals only Ph-positive cells (Dube et al., 1984a,b). Evidence that at least some Ph-negative cells that appear in long-term culture systems indeed derive from nonclonal stem cells was obtained by studying a patient with constitutional mosaicism for 46,XX and 45,X cells. The Ph-positive clone was associated with the minority 45,X karyotype. After 4-6 weeks of culture, metaphase cells were all 46,XX (Dube et al., 1 9 8 4 ~ ) . To summarize, Ph-positive CML is a disease characterized by clonal proliferation of pluripotent marrow stem cells. It probably progresses by multiple steps from a preleukemic Ph-negative stage through intermediate stages to blast crisis. Throughout much of the disease nonclonal stem cells persist in many, if not all patients, but their suppressed state is not influenced by conventional chemotherapy.

5. Ph-Negative CML Only two G6PD heterozygous patients with the less common disease Ph-negative CML have been studied (Fialkow et al., 1980; Kaye et al., 1984). Results support clonal involvement of a stem cell multipotent for erythrocytes and platelets as well as cells of the granulocytic series. The report of a patient with Ph-negative CML whose blast crisis cells bore multiple T-cell surface markers and stained cytochemically as immature T cells suggests the stem cell for this disease is pluripotent (Soda et al., 1985). These limited data indicate that

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although Ph-positive and Ph-negative CML differ clinically, they involve a similar stem cell. Recently, bcr, rearrangements have been found in leukemic cells from approximately one-third of patients with Ph-negative CML (Bartram and Carbonell, 1986; Morris et al., 1986). The unique restriction pattern in each case is consistent with a clonal nature of the disease. Of clinical significance is the observation that patients with bcr rearrangements (a molecular result of Ph) had disease whose clinical course was similar to that of the more indolent Ph-positive CML. It appears that at least a subset of patients without a cytogenetically detectable Ph chromosome in fact have Ph-positive disease. B. OTHERCHRONIC MYELOPROLIFERATIVE DISORDERS The chronic myeloproliferative disorders are a diverse group of diseases characterized by predominance of a subset of marrow elements: increased red cell mass in polycythemia Vera (PV); increases in megakaryocytes and platelets in essential thrombocythemia (ET); extensive marrow fibrosis with consequent marrow failure in agnogenic myeloid metaplasia (AMM). In myelodysplastic anemias the marrow shows dysfunctional maturation of cellular elements. Chromosomal abnormalities are often present and there is a predilection for progression to acute nonlymphocytic leukemia (ANLL) (Nowell, 1981, 1982). G6PD studies revealed that platelets, granulocytes, and erythrocytes from each of two patients with PV, three with AMM, and one with a myelodysplastic anemia exhibited the same single-enzyme phenotype, in contrast to normal tissue which contained both B- and A-type enzyme (Adamson et al., 1976; Kahn et al., 1975; Jacobson et al., 1978, 1982; Raskind et al., 1984; Fialkow et al., unpublished). Similarly, G6PD studies in five patients with ET heterozygous for GdBand either GdAor GdMedsupported clonal involvement of multipotent stem cells (Fialkow et al., 1981b; Gaetani et al., 1982). Since studies on cells from two of the patients with ET and one with PV were performed following treatment with 32P,cytoreductive therapy does not appear to return hemopoiesis to a normal nonclonal state. However, erythroid colonies and bursts grown with increasing concentrations of erythropoietin in vitro from two patients with PV showed an increasing proportion of bursts with the GGPD type not associated with the abnormally proliferating cells (Prchal et al., 1978). This observation suggests that normal stem cells persist but are suppressed in PV, as they are in CML. Of 117 independent EBV-transformed lymphoid lines expressing a

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single GGPD derived from one of the patients with PV, 108 expressed GGPD A, the type characteristic of the abnormal clone (Raskind et al., 1985). One hundred four of 109 GGPD-monotypic lymphoid lines established from one of the patients with ET and 135 of 148 monotypic lines from the patient with myelodysplastic anemia displayed the same enzyme type found in the clonally proliferating platelets and myeloid cells (Raskind et al., 1984, 1985, unpublished observations). These results indicate that PV, ET, and myelodysplastic anemia, like CML, involve a stem cell pluripotent for the lymphoid as well as the myeloid series. The cause of the marrow fibrosis in AMM is not known. However, GGPD studies indicate that it is not a primary result of the clonal proliferation. The observation that cultured marrow fibroblasts from two patients had a normal double-enzyme GGPD phenotype suggests that the marrow fibrosis is a secondary reactive phenomenon in AMM, as it is in CML (Jacobson et al., 1978; Fialkow et at., 1977, unpublished observations). Studies with GGPD were also informative with respect to the pathogenesis of a stable pancytopenia (Abkowitz et al., 1984). G6PD A and GGPD B were equally evident in skin and lymphocytes. Although there was no morphologic or cytogenetic evidence for preleukemia, the finding of a single GGPD type in erythrocytes, granulocytes, and platelets as well as in all cultured granulocyte/macrophage colonies and most erythroid bursts suggests an abnormal stem cell has clonally populated the marrow. Perhaps leukemia will later develop in this patient. However, it is also possible that the disordered hemopoiesis resulted from an exogenous injury that markedly depleted the stem cell pool. An abnormal or damaged clone resistant to the injurious agent might have emerged but might have been unable to support normal hemopoiesis. LEUKEMIAS (ANLL) C. ACUTENONLYMPHOCYTIC 1 . Evidence Supporting a Clonal DeveEopment

We have studied 21 patients heterozygous for GGPD who also had an acute myeloid leukemia (Wiggans et al., 1978; Fialkow et al., 1979, 1981a, unpublished; Fialkow and Singer, 1985; Jacobson et al., 1984). An additional 5 patients were reported by Ferraris et al. (1983, 1984). In all these patients blast cells exhibited a single-enzyme GGPD phenotype, indicating the clonal nature of diseases in this classification. Using a polymorphic restriction endonuclease site recognized by an HPRT probe as a cellular marker, Fearon et al. (1986) confirmed

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clonality of the blast cells in 7 of 10 patients heterozygous at that site. In 3 patients an anomalous blot pattern prevented interpretation.

2 . Dqferentiatiue Expression of the Stem Cells Involved in ANLL The differentiative expression of the stem cell producing the blast cells varies between patients. By examining GGPD expression in the different hemopoietic lineages a pattern of differentiation can be described. Since erythrocytes and platelets contain no DNA, RFLP marker systems cannot be used to investigate these lineages. In 13 relatively young patients with ANLL a pattern of restriction of differentiative expression largely to the granulocytic series was seenplatelets, erythrocytes, or both exhibited a double-enzyme phenotype (Fialkow et al., 1979, 1981a, unpublished; Fialkow and Singer, 1985). In contrast, five elderly women with ANLL were found to have a single GGPD enzyme expressed in all three lineages-granulocytic, erythroic, and megakaryocytic-consistent with involvement of a multipotent stem cell (Fialkow et al., 1981a, unpublished; Jacobson et al., 1984). In one of these latter patients evidence was obtained supporting the ability of the leukemic stem cell to differentiate to B lymphocytes (Ferraris et al., 1985b). That is, this patient’s disease involved a pluripotent stem cell, perhaps at the same developmental stage as the stem cell involved by CML. It is possible that the restricted differentiation pattern results when the leukemogenic event(s) occurs in a cell already committed to development along the granulocytic/monocytic pathway. Alternatively, an immature stem cell might be involved that is blocked from differentiation to other lineages. Using a variety of DNA probes, Fearon et al. (1986) were able to demonstrate that some mature granulocytes develop from the abnormal clone during periods of active disease. Two of six patients heterozygous for the HPRT RFLP had clonally derived circulating mature polymorphonuclear cells. In one patient whose leukemic blasts were trisomic for chromosome 8 and in another whose blasts were monosomic for chromosome 7 , densitometry of Southern blot bands obtained using probes for genes on those respective chromosomes detected a subset of granulocytes characterized by the same aneuploidies. In other words, the leukemic stem cell in some cases is able to produce fully mature granulocytic progeny, even during times of active disease and even if the stem cell is karyotypically abnormal.

3 . Remission Hemopoiesis in the majority of studied GGPD heterozygotes with ANLL became nonclonal during remission, as evidenced by a more

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balanced expression of both GGPD types (Fialkow et al., 1981a; Fialkow and Singer, 1985; Ferraris et at., 1983, 1984). However, in two patients whose leukemic stem cells showed multipotent differentiative ability there was persistence of clonal hemopoiesis in granulocytes and platelets as well as in granulocytic and erythroid colonies (Fialkow et al., 1981a; Jacobson et al., 1984; Fialkow and Singer, 1985). In one of these patients complex chromosomal rearrangements present during preceding and subsequent active disease phases were not apparent during the morphologic complete clinical remission. Although the pattern of differentiative expression could not be determined in studies using heterozygosity for an X-linked RFLP as a marker system, Fearon et al. (1986) found clonal remissions in 3 of 13 patients. The most likely explanation for these findings is that chemotherapy induced reversion to a clonal preleukemic state. Therefore, ANLL appears to be heterogeneous both by patterns of differentiative expression and by response to chemotherapy. It appears that patients whose disease is characterized by multipotent stem cell differentiative expression are less likely to achieve true remission. Perhaps this subset of patients, who tend to be older, have progressed through a long preleukemic prodrome during which clonal cells have almost totally replaced the normal marrow elements. The heterogeneity of ANLL has clinical significance: (1)in some patients morphologic and cytogenetic criteria may not be sufficient to diagnose remission; (2) different categories of ANLL may respond differently to antileukemic regimens. 4 , Multistep Pathogenesis The observation that clonal remissions are not rare in ANLL suggests that more than one step is required to transform a normal stem cell into a leukemic one. At least in some cases an intervening “preleukemic” phase probably exists. The patient reported by Jacobson et al. (1984) provides the strongest evidence for a multistep pathogenesis of ANLL. In this patient blast cells during acute disease were markedly abnormal cytogenetically, whereas the clonal cells present during clinical remission were cytogenetically normal. The clones in both stages of the disease were characterized by expression of the same GGPD type. It is likely that the aggressive leukemic stem cell derived from a clonally proliferating preleukemic stem cell. As in the case of overt ANLL, there is evidence for a multistep pathogenesis of a “preleukemic” condition, myelodysplastic refractory anemia. Two independent cytogenetically abnormal lines were found in marrow cells from a GGPD heterozygous patient with a myelodysplastic anemia 47,XX,+8 and 46,XX,del(1l)(q23), yet none

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of 14 lymphoid lines expressing the same G6PD allele as the myeloid clone contained these karyotypic alterations (Raskind et al., 1984). Just as was hypothesized for CML, the observations are consistent with existence of a stage in myelodysplasia during which there is clonal proliferation of genetically unstable but cytogenetically normal appearing stem cells. Subsequent development of specific chromosomal abnormalities confers selective advantage on subclones, and still additional alterations may lead to acute leukemia. V. Marrow Transplantation

A. RELAPSE FOLLOWING MARROWTRANSPLANTATION Allogeneic bone marrow transplantation has become an accepted therapy for some hemopoietic malignancies. The frequency of recurrence of leukemia after marrow transplantation varies with the type and stage of disease (Thomas, 1983). Although recurrences most often occur in host cells, occasionally donor cells may be involved. Since the first reported case of leukemia recurring in donor cells was published (Fialkow et al., 1971), at least nine more cases have been described (Thomas et al., 1972; Goh and Klemperer, 1977; Elfenbein et al., 1978; Cosset et al., 1979; Newburger et al., 1981; Schubach et al., 1982; Marmont et al., 1984; Witherspoon et al., 1985; Smith et al., 1985). Enzyme and DNA polymorphisms as well as sex chromosome differences have been used as markers to evaluate the origin of the cells in which the recurrences developed. Karyotype and/or Y body analysis in 54 sequential patients with acute leukemia who relapsed after receiving a marrow graft from a donor of opposite sex revealed that three cases (approximately 5%) developed malignancies in donor cells (two cases of ALL and one of immunoblastic sarcoma) (Boyd et al., 1982). These patients had been reported previously (Fialkow et al., 1971; Thomas et al., 1972; Schubach et al., 1982). An additional case of ANLL was suggested to be of donor origin by sex chromosome analysis (Goh and Klemperer, 1977). The presence of fluorescence banding polymorphisms allowed determination that one case of ALL and one of ANLL probably recurred in donor cells (Elfenbein et al., 1978; Newberger et al., 1981). A case in which immunoblastic lymphoma occurred following transplantation for ALL was investigated by DNA as well as by sex chromosome analysis (Schubach et al., 1982). Use was made of a probe from the highly polymorphic Ig heavy chain switch region that detected a

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different band pattern on Southern blots prepared from donor and recipient cells. Since the Southern blot pattern obtained from tumor cells with this Ig probe was identical to that seen in the donor, the sarcoma was considered to have arisen in donor cells. Data from Y body analysis were also consistent with this conclusion. Witherspoon et al. (1985) reported another case in which Ig RFLP differences between host and donor were used to evaluate a leukemia that recurred 6 years posttransplantation. Since donor and recipient were the same sex and no fluorescence banding differences were found, routine cytogenetic studies were not helpful in determining the origin of the leukemia. However, the finding that DNA from the recurrent leukemic marrow had the same Southern blot pattern for the Ig probe as did normal donor cells supports the conclusion that the recurrence originated in the engrafted donor cells. Lastly, one case of immunoblastic sarcoma occurring posttransplantation for ANLL was determined to be of donor origin by esterase D phenotyping (Gossett et al., 1979). The mechanism that initiates malignancy in the donor cells after marrow transplantation is not known. It is important to note that in none of these cases has leukemia or lymphoma been disgnosed in the donor. Among the possible explanations (Fialkow et al., 1971) is that a leukemogenic virus or other agent, such as a fragment of transforming DNA, is transferred to donor cells from radiation- or chemotherapydamaged host cells. Alternatively, some factor in the host environment may activate endogenous leukemogenic genes or viruses in the grafted cells. There have been two reports of leukemia recurring in donor cells following transplantation for Ph-positive CML. In one case, Ph was found in cells of the donor type sex (Marmont et al., 1984),whereas in the other case both karyotyping and Y body analysis revealed that the recurrent disease was present in donor-type cells without the Ph (Smith et al., 1985).As with the donor-cell recurrences of acute leukemia or lymphoma, the mechanism underlying these recurrences of CML are unclear. The increasing availability of RFLPs spanning the genome makes it increasingly likely that each host and donor of a transplant pair will be distinguishable on the basis of detectable DNA differences, with the notable exception of identical twins. A report by Blazar et al. (1985) suggests the utility of sequential DNA analysis in following the posttransplant course. In this study, at least one polymorphism was informative for each of 14 transplant pairs. As few as lo6 cells provided sufficient DNA for analysis, and in 3 cases this DNA method detected a minor population of 5-10%. The status of engraftment-none, par-

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tial, or complete-could be evaluated for unseparated marrow or for separated blood nucleated cell lineages. The advantage of this system is that host and donor cell populations can be distinguished even when they are matched for HLA and sex and even if there are no detectable enzyme protein or cytogenetic marker differences. Further studies on host cell recurrences may help elucidate the mechanisms by which leukemias arise de nouo.

B. MARROWSTROMAL MICROENVIRONMENT Marrow cells placed in long-term suspension culture develop an adherent cell layer that allows maintenance of in vitro hematopoiesis (Dexter et al., 1977). This adherent or “stromal” layer may be the in uitro counterpart of the marrow microenvironment required for hematopoiesis in uiuo. Adherent cells are a mixture of morphologically and antigenically distinct cell types, including fat cells, macrophages, and “endothelial-like” cells (Dexter et al., 1977; Keating et al., 1982). Transplantation between individuals who differ in enzyme phenotype or sex chromosomes allows the opportunity to investigate the differentiative potential of the transplanted pluripotent marrow stem cells. Cultured adherent layers from seven patients showed a direct correlation between time after transplant and percent donor-type cells by fluorescent Y-body assay (Keating et al., 1982). One patient was studied on two occasions. At day 14 posttransplant, 40% of the adherent cells displayed a fluorescent Y body; at day 86, the Y body could be detected in 67% of stromal cells. The adherent layers from two patients studied more than 1year posttransplant were entirely donor in origin by this technique. These results demonstrate the transplantability of the cell(s) responsible for in uitro adherent (stromal) cells and suggest that there might exist a common progenitor for marrow hemopoietic and microenvironmental cells. Morphologic and antigenic evaluations have shown that fibroblasts comprise only a minor component of the adherent layer in humans (Keating et al., 1982; Singer et al., 1983). In contrast to the majority of adherent cells, cultured marrow fibroblasts apparently are not transplantable (Golde et al., 1980). Studies with G6PD heterozygous patients having clonal neoplastic diseases have also supported the nonidentity of marrow stroma and marrow fibroblasts. The stromal layer set down during long-term culture by marrow from a patient with Phpositive CML displayed the same monomorphic G6PD phenotype as her malignant myeloid cells, yet marrow fibroblasts showed both A and B enzymes (Singer et al., 1984).

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Studies in patients with clonal hematopoietic diseases provide evidence that some in vitro adherent cells differentiate from the hematopoietic stem cell involved by the diseases (Singer et al., 1984). The adherent layer that developed in vitro from each of four GdB/GdA heterozygotes-one with Ph-positive CML, one with Ph-negative CML, and two with ANLL-manifested a single GGPD type, identical to that found in the tumor cells. The involved stem cell in each of these patients was capable of differentiation to erythroid, myeloid, and megakaryocytic cells since these uncultured fractions as well as cultured BFU-E and CFU-C shared clonal GGPD expression with the tumor cells. In contrast, the adherent layer from a patient with unipotent-differentiative-expression-type ANLL was found to produce both GGPD A and B in equal amounts. The finding in myeloproliferative diseases involving multipotent stem cells that some in vitro adherent cells are probably derived from the same progenitors as the neoplastic cells suggests that hemopoietic and stromal elements may share a common stem cell in normal hematopoiesis as well.

VI. Concluding Remarks

Over the past two decades studies utilizing cell marker systems have been instrumental in establishing that monoclonality of hemopoietic tumors is the rule, not the exception. Additionally, the ability to distinguish the tumor cell population from coexisting normal cells has enabled researchers to follow these cellular compartments through the course of disease. Knowledge of the response of leukemic cells to chemotherapy will be important in devising therapeutic regimens. For example, the finding of leukemic cells that persist during periods of clinical remission may identify patients whose prognosis is poor with standard therapy. Many human hemopoietic neoplasias appear to have a multistep pathogenesis. The diploid cell may represent an early stage in leukemogenesis. Subsequent development of structural chromosomal alterations or aneuploidies may lead to frank leukemia by any of a number of mechanisms, e.g., activation of an oncogene, amplification of a gene whose product is important for growth, or loss of a gene involved in repressing or regulating growth. The malignant phenotype then progresses by stepwise evolution. Future endeavors to define these various oncogenic steps may provide important information of clinical as well as biological significance.

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ACKNOWLEDGMENT The authors’ studies were supported by Public Health Service Grant Number CA 16448 awarded by the National Cancer Institute, Department of Health and Human Services.

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MULTISTAGE MODEL OF NATURAL KILLER CELL-MEDIATED CYTOTOXICITY INVOLVING NKCF AS SOLUBLE CYTOTOXIC MEDIATORS Benjamin Bonavida and Susan C. Wright Department of Microbiology and Immunology. UCLA School of Medicine. Universify of California at Los Angeles. Los Angeles, California 90024

I. Introduction

It has been over a decade since the first description of the existence of a subpopulation of lymphocytes termed natural killer (NK)' cells (Takasugi et aZ., 1973; Rosenberg et aZ., 1974; De Vries et al., 1974; Kiessling et aZ., 1975). Although not totally satisfactorily, NK cells have been defined functionally as null cells (non T, non B, nonmacrophage, neutrophils, or granulocytes) that have the ability to lyse certain target cells in a non-MHC-restricted manner and in the absence of prior sensitization. Several surface markers associated with NK ceIls have been identified by monocIona1antibodies such as HNK1 (Abo and Balch, 1981), N901 (Griffin et al., 1983), and B73.1 (leu 11) (Perussia et aZ., 1983). There is a large body of evidence supporting the notion that NK cells play an important role in host defense mechanisms against primary and metastatic tumors and viral infections (Warner and Dennert, 1982; Heberman, 1983; Hanna, 1983).Also, NK cells have been implicated in the regulation of hemopoiesis and immune responses (Abbruzzo and Rowley, 1983). The molecular mechanism of NK CMC resulting in recognition and target cell lysis remains unknown though several postulated mechanisms have been suggested. Little is known about the nature of the NK recognition structure, the target cell structure for recognition, adhesion molecules, signal transduction, the lethal hit, how target cells are lysed, and what distinguishes resistant versus sensitive targets. 1 Abbreviations used: NK, natural killer; MHC, major histocompatibility complex; CMC, cell-mediated cytotoxicity; CTL, cytotoxic T lymphocytes; NKCF, natural killer

cytotoxic factors; PBL, peripheral blood leukocytes; rLT, recombinant lymphotoxin; rTNF, recombinant tumor necrosis factor; IL-2, interleukin-2; LGL, large granular lymphocytes.

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These and other questions are currently under investigation in several laboratories, and progress has been made in all of the above areas. This was in part due to previous findings that have examined the mechanism of CTL-mediated cytotoxicity and the similarities that have been found between the mechanisms of NK- and CTL-mediated lysis. II. Postulated Mechanisms of the NK CMC Reaction

Several studies have investigated the characteristics of the NK cytotoxic system and its relationship to CTL-mediated cytotoxicity. In such studies, the assays used are essentially similar and most notable are the W r release assay (Brunner et al., 1968), the single cell cytotoxic assay (Grimm and Bonavida, 1977; Targan et al., 1980), and the dye reduction assay (Callewaert et al., 1982). It has been established that NK-tumor target interaction proceeds through several discrete steps, namely, (1)effector target cell recognition and binding, (2) programming for lysis or the lethal hit, and (3)target cell lysis independent of the NK effector cell (Roder and Lauzon, 1983; Hiserodt et al., 1982a; Kahle et al., 1983). Roder and colleagues (1981) have postulated a “stimulus secretion model of NK cytolysis” in which the target cell stimulates the NK effector cell to release a cytolytic factor which then binds to and lyses the sensitive target cells (reviewed by Kiyohara et al., 1986). The first demonstration of a specific soluble cytotoxic factor produced by NK cells and implicated in the NK CMC reaction has been reported by Wright and Bonavida (1981, 1982) and Wright et al. (1983). These NK cytotoxic factors (NKCF) have been examined for their role in the NK CMC reaction and found to exhibit a good correlation with the known characteristics of NK cells (Bonavida and Wright, 1986a,b; Wright and Bonavida, 1986a). Other investigators have reported the presence of cytoIytic granules in NK clones and tumors and implicated them in the mechanism of NK CMC (Dennert and Podack, 1983; Podack and Dennert, 1983; Millard et al., 1984). This article focuses primarily on accumulated evidence supporting the role of NKCF in NK cytotoxicity and briefly compares the characteristics of NKCF with other cytotoxins postulated to be involved in CMC reactions. 111. Multistage Model of the Mechanism of NK CMC and Involving NKCF as Soluble Cytotoxic Mediators

Depicted schematically in Fig. 1 is our proposed model for the mechanism of NK CMC. The first step of the lytic pathway involves the interaction of a “receptor” (I) on the NK cell which recognizes and

FIG. 1. Model proposed for natural killer cell-mediated cytotoxicity. I, NK recognition structure; 11, NK target structure(s);111, stimulating target cell structure; IV, “receptor” for activation of NK effector; v, secretory apparatus;VI, release of NKCF; VII, NKCF binding site; VIII, NKCF processing; IX; target-cell death.

binds to the NK target structure (11)on the cell membrane. The effector cell must then receive a signal from the target to activate the NKCF release mechanism. This is depicted as occurring through the interaction of target cell membrane determinants (111) with corresponding “receptors” (IV) on the effector cell, and both interactions are thought to be distinct from the initial binding step. Subsequent to stimulation of the effector cell, the secretory apparatus (V) is activated to release NKCF (VI) in close proximity to the target cell. Following release, NKCF bind to NKCF binding sites (VII) which may then be processed (e.g., internalized) (VIII), resulting ultimately in irreversible target cell death (IX). IV. NKCF as Soluble Mediators in NK CMC

In our initial studies, we postulated that coculture of NK cells with target cells should result in the secretion in the supernatant of soluble cytotoxic mediators that are selectively cytotoxic to NK-sensitive target cells. Indeed, we were able to demonstrate that NKCF are produced following culture of effector cells and target cells for 20-26 hr in the absence of serum (Wright and Bonavida, 1981). The NKCF were selectively lytic for NK-sensitive target cells and also species specific when assayed in an 18-to 20-hr 51Crrelease assay. Production of NKCF in the human system is mediated by plastic nonadherent PBL whereas for mice and rats plastic nonadherent spleen cells are employed. The cell types that release NKCF were determined. In the murine system, NKCF are produced by spleen cells but not thymocytes, and do not require the presence of mature T cells or adherent cells (Wright and Bonavida, 1982). In man, the NK-enriched LGL fraction of PBL

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release NKCF (Wright et al., 1983), and NK cells purified by selection of Fc receptor-positive LGL also produce NKCF (Degliatoni et al., 1985). These data are consistent with the NK effector cell producing NKCF. V. Evidence Supporting the Multistage Model in the Mechanism of the NK CMC Reaction

A. RECOGNITION AND BINDINGOF NK CELLS TO TARGET CELLS

The first step in the NK CMC reaction is target cell recognition by the NK cell, and, in conjunction with cell adhesion molecules, binding between the two cells takes place. The nature of the putative NK recognition receptor is not yet known. Studies done with fresh PBL failed to reveal the presence of gene rearrangement for the p chain of the antigen-specific T cell receptor (Lanier et al., 1986). In contrast to fresh PBL, NK-like clones in uitro have been shown to rearrange and express the p chain gene product (Yanagi, 1985). However, it is not clear whether these clones are indeed derived from N K cells or are T cells that express NK-like activity. The clonal distribution of recognition receptors on NK cells have been primarily derived from cloned human NK cell lines displaying heterogeneous and discriminate cytotoxic activity (Hercend, 1983). This, however, could be the result of one or more factors that affect target cell susceptibility to lysis (see below) and could be unrelated to the effector cell recognition apparatus. Several laboratories have invested effort into the isolation and characterization of NK target cell structures (Roder et d.,1979a,b). Three molecular weight species of 130,000,160,000, and 240,000 were identified on a murine NK-sensitive target cell. It appears that multiple target cell specificities exist, based on indirect techniques (Phillips et al., 1980; Jensen and Koren, 1979; Callewaert et al., 1979). The transferrin receptor and viral glycoproteins have been suggested as candidates for NK target structures (Vodinelich et d . , 1983; AbendrothBishop et al., 1983). Also, genetic factors have been implicated in the expression of NK target structure (Ahrlund-Richter et al., 1980; Clark, 1986).However, the exact role of such genetic factors in NK CMC may be assigned to one or more stages of the NK CMC reaction as diagrammed in Fig. 1. Here again, the complexity of the system and the presence of several cell adhesion molecules on both the effector and target make it difficult to ascertain which structures are involved and

whether the “affinity” of interaction between NK and target cells may explain several of the results observed. Clearly, in efforts to resolve such complex problems, it is essential to characterize biochemically the molecules involved in binding or recognition and then assess their role and function. The interaction of an NK effector with a target results in the formation of conjugates that are visible microscopically. This binding is dependent on the cation Mg2+. Binding in the absence of Ca2+ inhibits the subsequent programming phase for lysis and no lysis takes place. This ability to discriminate between binding and postbinding events is a technique useful in investigating the role of various inhibitors in preventing conjugate formation or postconjugate formation. For example, a blocking antibody was reported to inhibit conjugation formation at the binding level of the effector cell (Newman, 1982; Kahle et al., 1983). Other soluble inhibitors were also reported that can inhibit binding or postbinding events (Roder et aZ., 1981).

B. ACTIVATIONOF

THE

NKCF RELEASEMECHANISM

The state of the lipid bilayer and the organization of the cytoskeleton have been shown to be important for NK cytolysis (Roder et al., 1978, 1980). Methylation of phospholipid is required for cytolysis (Hoffman et al., 1981), and it was also shown that transmethylation may be involved in the triggering stage before Ca2+-dependentprogramming for lysis (Quan et d.,1982). Calcium is required in cytolysis by NK cells. The site of action of Ca2+ is not clear. It has been suggested that calmodulin may be involved in regulating Ca2+ATPase activity associated with phospholipid methylation in cell membranes. Calmodulin inhibitors all inhibited NK cytolysis (Quan et al., 1982; Moon et d.,1983). In the absence of inhibitors of binding, several cyclic nucleotides regulate cytotoxicity at the postbinding phase (Katz et al., 1982).The lysosomal enzyme, chloroquine, inhibits the lytic phase of NK cytotoxicity when the effector cells were pretreated, suggesting that some lysosomal elements are important in NK lysis (Quan et aZ., 1982). Following the initial recognitionhinding event between the NK effector and the target, the target must be able to stimulate the NKCF release mechanism. Binding alone is not sufficient to activate the NKCF release mechanism as some target cells bind to NK cells but do not stimulate NKCF release. For instance, target cells pretreated with interferon (IFN) bind to NK cells but do not stimulate NKCF production (Wright and Bonavida, 1983b), thus accounting for their resis-

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tance in the NK CMC reaction (Trinchieri et al., 1978). Therefore, we have proposed that following the initial recognitiodbinding event a second signal is transmitted to the NK cell through the interaction of the same set of receptors or, alternatively, through a completely different set of membrane determinants. The molecular nature of such ligand-receptor interactions is not known. We have demonstrated that NK cells can be triggered for release of NKCF by mitogens such as ConA and PHA or by the action of TPA and Ca2+ionophore (Graves et al., 1986). It is possible that target cell stimulation of NKCF release follows the same pathway of activation as that mediated by TPA and ionophore. Recent evidence from our laboratory indicates that protein kinase C and increased cytosolic Ca2+are involved in the initiation process for secretion of NKCF. Both TPA and Ca2+ionophore are required, and the activation pathway is consistent with the events following effector-stimulus interaction leading to metabolism of the membrane lipid phosphatidylinositol bisphosphate to inositol triphosphate and lY2-diaglycerol.This, in turn, leads to release of Ca2+ into the cytoplasm from the endoplasmic reticulum store, activation of protein kinase C, and protein phosphorylation. Several reports have presented evidence that proteolytic enzymes are involved in the NK lytic mechanism (Quan et al., 1982; Roder et al., 1978; Hudig et al., 1981; Goldfarb et al., 1982; Lavie, 1982). These studies were in large part based on the ability of various protease inhibitors to inhibit the NK CMC reaction. Since these inhibitors did not inhibit the initial recognitionbinding event, it has been suggested that they block the postbinding or lethal hit event of the NK cytolytic pathway. The role of protease inhibitors was examined in the NKCF system. Based on the proposed model shown in Fig. 1, proteases could be involved in either the NKCF release mechanism or target cell lysis by NKCF. These possibilities were tested using inhibitors shown previously to inhibit NK CMC. Inhibitors of serine-dependent proteases and/or sulfhydryl-dependent enzymes inhibited the production of NKCF by NK cells stimulated with target cells. These same inhibitors had no effect on target cell lysis by NKCF (Wright and Bonavida, 1984a). These findings indicated that the relevant proteases may be associated with the effector cell surface or else may be intracellular since the low molecular weight inhibitors could presumably pass through the cell membrane. Also possible, the putative enzymes may be involved in the NKCF release mechanism, or else it may process NKCF precursors into lytically active forms. Thus, inhibition

of production of lytically active NKCF could account for the observed inhibition of NK CMC by protease inhibitors. OF NKCF C. SECRETION

1 . Evidence for Secretion in N K CMC There are several lines of compelling evidence that secretion is involved in the mechanism of NK CMC. Monensin is a carboxylic acid ionophore that causes an accumulation of cytoplasmic vesicles in LGL and inhibits NK cytolysis (Carper et al., 1981).Since monensin interrupts the vesicular traffic of Golgi-derived vacuoles to the cell surface, it is postulated that Golgi-derived products may be carried to the NK cell surface for delivery to the target cells. Degranulation of LGL with S?+ results in consistent loss of cytolytic activity (Neibour and Huberman, 1982). Verapamil, a Ca2+channel blocker, inhibits secretion and cytolysis, demonstrating that a Ca2+flux across the membrane is necessary (Hiserodt et al., 1982b). Reagents that inhibit cytolysis (Roder and Klein, 1979) also block lysosome secretion in other cells (Weissman et al., 1975), and agents that increase lysosomal discharge augment NK cytolysis (Roder and Klein, 1979; Katz et al., 1982). These various agents were also shown to inhibit NKCF production, thus corroborating the role of NKCF secretion in NK CMC. 2 . Kinetics of Secretion of N K C F In the NK CMC reaction, the lethal hit event and target cell death occur rapidly within 1-3 hr of contact between the NK and target cell. Accordingly, if NKCF play a role in NK CMC as proposed, sufficient amounts of NKCF should be released within a short time following stimulation of the NK effector by the target cell. However, our studies on the induction of NKCF reveal that maximal activity is obtained after 11-20 hr incubation of effector and stimulator targets (Wright and Bonavida, 1982). The kinetic differences seen between the NK CMC reaction and NKCF production may be due to several possibilities, namely, the insensitivity of the assay for detecting small amounts of NKCF, the instability of NKCF, or presence of inhibitors, or may be due to the optimal delivery system of NKCF onto the target cell in the NK CMC reaction. We first examined whether the NK effector cells are capable of releasing NKCF in a short time. The effects of mitogens such as ConA and phorbol ester and ionophores were examined. The findings of

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such experiments indicate that NKCF production can take place rapidly within 30 min following stimulation of NK cells with either Con A or TPA and ionomycin (Graves et al., 1986). These studies suggest that the NK effector cells could release NKCF within a time commensurate with the kinetics of NK CMC and, therefore, corroborate our hypothesis that NKCF play a role in NK CMC. 3. Kinetics of Cytotoxicity by NKCF During the NK CMC reaction, target cell lysis occurs rapidly in 1-4 hr as assessed in the 51Crrelease assay. Therefore, if NKCF play a role in NK CMC, target cell lysis by NKCF must also take place rapidly, However, under standard conditions, lysis of sensitive target cells by NKCF requires 16-20 hr incubation (Wright and Bonavida, 1984b). Several possible mechanisms may be responsible for this discrepancy. For instance, (1) the NKCF released in the supernatant following coculture of NK and target cells may become partially inactivated or else too diluted to mediate rapid target cell lysis, (2) it may be that there exists a specialized NKCF delivery mechanism operating when NK cells are in direct contact with the target and facilitating target cell lysis, (3) it is possible that other cofactors play a role in NKCF-mediated lysis of target cells in the NK CMC reaction. In fact, we have observed that target cells treated with protein synthesis inhibitors are more susceptible to lysis by NKCF and the kinetics of lysis are enhanced so that lysis can be observed within 4-6 hr, a time compatible with NK CMC reaction (B. Bonavida and S. C. Wright, unpublished). These studies suggest that NKCF can lyse target cells in a short-term assay and support its role as soluble mediators in the NK CMC reaction. D. BINDINGOF NKCF

TO

NKCF BINDINGSITESON TARGET CELLS

We have shown that coculture of NK cells with targets results in the production of NKCF, and NKCF can lyse NK-sensitive target cells. The interaction of NKCF with target cells may occur through NKCF binding sites or receptors or through other mechanisms not involving specific receptors. Several lines of investigation were unable to distinguish between these possibilities, and the results suggest strongly that target cells bear NKCF binding sites to which NKCF can bind during the initial phase of the cytotoxic reaction. Evidence in support of the presence of NKCF binding sites on target cells emanate from different lines of experimentation as summarized:

1. Several NK-sensitive and NK-resistant target cells adsorb NKCF from supernatants containing NKCF. The adsorption is dependent on the temperature used, time of adsorption, and number of cells used for adsorption (Wright and Bonavida, 1983a; Wright et al., 1986). Cells lacking NKCF binding sites and resistant to NK CMC were also resistant to NKCF-mediated lysis. The finding that both NK-sensitive and -resistant target cells can adsorb NKCF suggested that the presence of NKCF binding sites on target cells is required but not sufficient for NKCF or NK CMC-mediated target cell lysis. 2. Adsorption, as well as target cell lysis by NKCF, can be prevented by the addition of the same monosaccharides (Wright and Bonavida, 1981) shown previously to inhibit NK CMC in the human (Forbes et al., 1981) or murine systems (Stutman et al., 1980). These findings suggested that carbohydrate determinants expressed on either NKCF molecules or target cell binding sites are essential for their interaction leading to target cell lysis in the NK CMC reaction. Alternatively, the relevant carbohydrate determinants may be expressed on the binding site since tunicamycin-treated target celIs became relatively resistant to lysis by either NKCF or NK cells (Blanca et al., 1985). 3. Further evidence for the role of NKCF binding sites in the NK lytic mechanism is derived from studies using enzyme-treated target cells. Following treatment with papain, the target cells became insensitive to the lytic effect of NKCF, were resistant to NK cells, and did not adsorb NKCF. These treated target cells, nevertheless, formed conjugates with NK effector cells (Wright et al., 1986). These results suggested that the NKCF binding sites on target cells are papain sensitive and that the expression of protein-containing structures required for conjugate formation is required for NK sensitivity. 4. The hypothesis that the expression of NKCF binding sites on the target cell is essential for target cell NK sensitivity was tested by developing NKCF-resistant variants derived from the NK-sensitive YAC-1 and U937 target cells. Prolonged culture of such targets in NKCF-containing medium resulted in the development of NK-resistant variants (Wright and Bonavida, 1983a, Wright and Bonavida, 1986b). These variants adsorbed NKCF poorly. However, they form conjugates, stimulate NKCF release, but were resistant to lysis by NKCF, and thus account for their NK resistance. These findings demonstrate directly the important role of NKCF binding sites in the NK CMC reation. Also, these studies indicate that the NKCF binding site is distinct from those membrane structures involved in the first two

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steps of the cytolytic reaction, namely, the recognitiontbinding and stimulation of release of NKCF.

E. EVENTS FOLLOWING BINDING OF NKCF TO TARGET CELLS In the previous section, we have indicated that the interaction of NKCF with binding sites on target cells is required for lysis; however, NKCF binding alone is not sufficient to lyse the target cell. For instance, YAC-1 cells rendered NK resistant by passage in vivo are resistant to lysis by NKCF even though they adsorb NKCF as well as the NK-sensitive YAC-1 cells cultured in vitro (B. Bonavida and S. C. Wright, unpublished observations). Thus, NKCF postbinding events are essential to bring about cell lysis. The involvement of proteolytic activity was not readily apparent since various types of anti-proteases had no effect on target cell lysis by NKCF (Wright and Bonavida, 1984a). We investigated whether NK-resistant targets could be lysed following fusion with liposome-encapsulated NKCF. Preliminary studies suggest that NK-resistant targets are lysed following fusion with liposome-encapsulated NKCF (Roozemond et al., 1986a). In these studies it is not clear whether the liposome fusion allows the delivery of NKCF into the cytoplasm and whether other intermediate steps are involved. Other recent studies indicate that NK resistance to NK CMC is a property of the membrane as fusion with liposomes containing membranes from NK-sensitive targets render NK-resistant targets sensitive to lysis by NK cells in NK CMC (Roozemond et al., 198613). Using time-lapse cinematography, we have observed morphological changes in target cells incubated with NKCF. Within 1-2 hr after exposure to NKCF, the target cells tend to clump up followed by a period of membrane blebbing and zeiosis which just precedes cell death (B. Bonavida and S. C. Wright, unpublished). Overall, it is not yet clear how NKCF mediate lysis of target cells, and the biochemical pathways involved are not yet identified. VI. Properties Which Determine NK Sensitivity and Resistance

In our various studies we have made use of a different number of NK-resistant variants as means to dissect the various stages of the mechanism of NK CMC as depicted in Fig. 1.Based on these studies, we determined the minimal requirements for a target cell to be NK sensitive, and these include (1)the ability to be recognized and bound by the NK effector cell, (2) the ability to stimulate the NKCF release

mechanism, (3)the expression of NKCF binding sites to which NKCF can bind, and (4)processing of NKCF by as yet unknown mechanisms leading ultimately to cell lysis. Each of these various steps could be studied independently of each other. Thus, in the first step, treatment of target cells with trypsin inhibits their ability to bind to NK cells and, therefore, acquire resistance to NK cells. It is not known yet whether such cells are sensitive to NKCF. The second step of triggering the NKCF release mechanism was found to be defective when the target cell was pretreated with IFN or when the cultured NK-sensitive YAC-1 target was passaged in ascites form in vivo. While the IFNtreated target was sensitive to NKCF-mediated lysis, the YAC-1 ascites form was not. Culture of NK-sensitive targets in NKCF-containing supernatants or treatment with papain renders the cells insensitive to NKCF and NK presumably because of loss of NKCF binding receptors. Therefore, the ability to identify each of the required properties for NK sensitivity for every new target cell tested permits the precise delineation of the block(s) responsible for target cell resistance to NK cells. Thus, NK-resistant tumor cells or virally infected cells may exhibit different escape mechanisms and may require different manipulations to render them NK sensitive. Two approaches were investigated to determine whether NK-resistant target cells can be rendered NK sensitive. The first approach investigated whether the sensitivity to NK lysis is a property of the membrane of the target cell. This was analyzed by the application of cell-liposome fusion. Our recent studies demonstrated that NK-resistant tumor cells acquired sensitivity to lysis by NK cells after fusion with reconstituted vesicles containing membrane components derived from NK-sensitive target cells. This finding was demonstrated in both the human and murine systems. The conversion to NK sensitivity was specific as target cells were not lysed when fused with vesicles containing membrane components derived from either NK-resistant targets or NK-sensitive targets of another species (Roozemond et al., 198613). These results demonstrate the feasibility of converting a resistant NK target to a sensitive target by cell-liposome fusion and the possibility of applying this technique in clinical settings. The second approach investigated whether target cells resistant to NKCF can be rendered sensitive if fused with liposome-encapsulated NKCF. Our preliminary studies indicate that NK-resistant targets (lacking NKCF binding sites or ability to process bound NKCF) can be lysed following fusion with liposome-encapsulated NKCF (Graves et al., 1986; Roozemond et al., 1986a). These results indicate that

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resistance to NK or NKCF can be overcome, but sensitivity to these manipulations remain a property of the target cell under study. VII. Biochemical Characterization of NKCF

Several studies have been undertaken to characterize biochemically the properties of NKCF. Although several properties were determined, to date purification of NKCF to homogeneity has not been achieved. Nevertheless, considerable information has been accumulated on the characteristics of NKCF (Wright et al., 1985).NKCF activity is stable at 4°C and freezing, unstable at 63"C, 8 M urea, or pH 2. These factors are sensitive to trypsin, and NKCF activity is eliminated by reduction and alkylation. These results suggested that NKCF are proteins in which disulfide bonds are essential for lytic activity. Mild oxidation with sodium periodate inactivates NKCF, suggesting that carbohydrate determinants are essential for lytic activity. NKCF from different species migrate in a single band peak exhibiting an apparent MW of 15,000-40,000 by HPLC gel filtration. This molecular weight range corresponds to that reported by other investigators for human NKCF (Degliantoni et al., 1985; Blanca et al., 1985). In preliminary experiments, different fractionation techniques revealed that NKCF consists of more than one type of cytotoxic molecule, and ongoing studies are directed at efforts to purify NKCF and correlate its activity with other cytotoxic molecules. VIII. Relationship of NKCF to Other Cytotoxins

In addition to NKCF, several candidates as soluble mediators in cytotoxicity have been described, most notably lymphotoxin (LT), tumor necrosis factor (TNF), cytolytic granules, and their cytolytic products cytolysin or perforins. Since NKCF have not yet been purified to homogeneity, it is not clear how these factors relate to these other cytotoxic molecules. However, it is possible to make comparisons based on functional studies, and such studies were performed with the recently available recombinant LT and TNF.

A.

RELATIONSHIP BETWEEN

NKCF AND rLT

Lymphotoxin (LT) is a cytotoxic factor derived from activated lymphoid cells that has cytostatic and cytotoxic activity against tumor cells (Williams and Granger, 1968; Ruddle and Waksman, 1968). LT has been implicated as the mediator of several types of CMC reactions

(Walker and Lucas, 1973; Ware and Granger, 1981; Schmid et al., 1985; Kondo et al., 1981), as well as in NK CMC (Weitzen et al., 1983a,b). Various forms of LT have been described ranging in MW from 12,000 to 200,000 (Granger et al., 1978). Recently, LT has been biochemically purified (Aggarwal et al., 1984, 198513) and cloned in Escherichia coli (Gray et al., 1984). We have undertaken a functional comparison between human NKCF and purified recombinant LT (rLT), generously donated by Genentech. We found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF (Wright and Bonavida, 1987). B. RELATIONSHIP BETWEEN NKCF AND rTNF Tumor necrosis factor (TNF) is another cytotoxic mediator originally described by Carswell et al. (1975). TNF is primarily produced by monocytes and macrophages (Mannel et al., 1980; Nissen-Meyer and Hammerstrom, 1982). TNF has been implicated as a mediator of adherent cell-mediated cytotoxicity (Zascharchuk et al., 1983; Mathews, 1983).Recently, TNF has been biochemically purified (Aggarwal et al., 1985a,b) and its gene expressed in E . coli (Pennica et al., 1984; Shirai et al., 1985; Wang et al., 1985). A comparison between NKCF and rTNF was recently made. TNF was found to exhibit in large part a target cell specificity similar to NKCF. Furthermore, antibodies directed against TNF mediated partial to complete inhibition of lysis of U937 target cells by NKCF. These results suggested that NKCF was the same as or antigenically similar to TNF (Wright and Bonavida, 1987). The role of TNF in NK CMC was tested in antibody blocking experiments. Anti-TNF caused partial inhibition of NK CMC, and therefore it was not possible to determine whether TNF plays a role in NK CMC. To analyze further the possible role of TNF in the NK lytic mechanism, we developed variants by the prolonged culture of U937 in the presence of TNF. Variants selected by TNF (u937TR) were compared to variants selected by growth in NKCF (u937NR). It was found that both variants were resistant to lysis by either TNF or NKCF. However, only U937NRand not U 9 3 7 were ~ ~ found resistant to Iysis by fresh or activated NK cells. Other comparative studies are shown in Table I. Since TNF could not induce NK resistance, this suggests that TNF alone is not sufficient to mediate NK activity in a

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TABLE I COMPARISON OF TARGET CELLVARIANTS SELECTED FOR RESISTANCE TO TNF OR NKCF Target cella Characteristics General properties Conjugate formation Stimulation of NKCF Adsorption of NKCF Binding of rTNF Susceptibility to lysis by NK CMC Antibody-dependent cellular cytotoxicity Lectin-dependent cellular cytotoxicity IFN-activated macrophage NKCF rTNF

u937

U937TR

+ + + +

+ + ? +

+

+

+

+

+

u937NR

+

+

+ + + ~~

The u937TR (TNF resistant) and u937NR (NKCF resistant) target cells were prepared by culture of the susceptible U937 target cell in medium containing rTNF or NKCF, respectively. The resistant variants were cloned and subcloned prior to testing.

CMC reaction. It is possible that NK CMC is mediated by a composite of cytotoxic factors which are all components of NKCF. Further biochemical analysis of NKCF should clarify their relationship to TNF and their role in the NK lytic mechanism. C. RELATIONSHIP BETWEEN NKCF AND CYTOLYSIN OR PERFORINS Cytolysins or perforins have been implicated as mediators of NKand CTL-mediated cytotoxic reactions (Podack and Dennert, 1983; Millard et al., 1984). These molecules have been isolated from the granules of various target cell lines and are proposed to lyse target cells by forming pores in the membrane. These molecules lyse target cells very rapidly, are not species or target cell specific, and lyse cells at room temperature. Such properties are not shared by NKCF; therefore, we postulate that NKCF are different from cytolysins or perforins. However, direct comparison and antibody-mediated blocking experiments are required for further characterization.

IX. Clinical implications of the NKCF System

Numerous studies have provided evidence that NK cells play an important immunoregulatory role as well as a role in defense against infection and neoplasia. Therefore, a detailed understanding of the NK effector system and its mode of action is important to detect abnormalities that may arise and means to restore their function. Our studies presented above have analyzed the NK cytotoxic system and established several stages of the lytic pathway that can each be analyzed and assessed independently. This staging system permits the precise delineation of the block(s) that may be responsible for a defective NK cytotoxic system. Once the block(s) is identified, one may evaluate means to correct the defect and means to follow the restoration. Several factors may be implicated in a defective NK system or in inherent resistance of target cells to lysis by NK cells. Such factors are listed in Table 11. Below, we provide one example of the clinical application of the NKCF system in patients with AIDS. Peripheral blood from AIDS patients exhibits low or no NK activity. The mechanism underlying the defective NK activity was examined in our laboratory (Bonavida et al., 1986). Following the scheme proposed for the mechanism of NK CMC, we made the following observations: (1) The frequency of NK cells capable of binding and forming TABLE I1 FACTORS IMPLICATED IN A DEFECTIVE NK SYSTEM OR IN INHERENT RESISTANCEOF TARGET CELLSTO LYSISBY NK CELLS Possible mechanisms resulting in a defective NK cytotoxic activity Reduced frequency of NK cells Failure of precursor cells to differentiate into mature NK cells Expression of low affinity target recognition structures and/or adhesion molecules Deficiency in lymphokine (IL-2, IFN) production required for NK cell regulation and maturation Defective trigger for the stimulation of NKCF Defective secretory apparatus Inability to synthesize NKCF Production of inactive NKCF or secretion of inhibitors of NKCF Factors responsible for target cell resistance to NK-mediated cytotoxicity Lack of target cell structures recognized by NK cells Absence of or low affinity trigger structures responsible for activation of the NKCF release mechanism Absence of low affinity, or defective NKCF binding sites Failure to process bound NKCF for lysis Elevated repair mechanisms

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conjugates with NK-sensitive targets was normal. This suggested that NK cells are not depleted in AIDS patients and also that the recognition structures on NK cells are not defective. (2) In contrast to normal cells, AIDS NK cells failed to release NKCF following stimulation with target cells. This localized the defect at the trigger responsible for activation, the NKCF release mechanism, or the failure to synthesize NKCF. (3) AIDS NK cells, however, responded to stimulation with mitogenic ConA and released NKCF in the supernatants, albeit at lower levels than controls. These results demonstrated that AIDS NK cells synthesize NKCF and secrete NKCF by appropriate stimulation. They appear defective in the trigger receptor responsive to the target cell stimulation. (4) The trigger, however, is not totally defective as cells pretreated with IL-2 respond to target cell stimulation and release NKCF. These results suggested that the defective trigger can be up regulated by IL-2. Since IL-2 production is defective in AIDS patients, these studies suggest that the trigger for NKCF release may be under IL-2 regulation. Clearly, the studies done with AIDS patients can be extended to other diseases where the NK system is deficient. X. Remarks

This article on the NKCF system covers mainly areas of research undertaken in the authors’ laboratory. The intent was not to review the literature on the NK system but to merely focus on the possible mechanism of NK CMC in which the soluble cytotoxic mediators NKCF are implicated. Clearly, the studies proposed here for the NK cytotoxic system may be applicable to other cytotoxic systems mediated by non NK effector cells. Although functionally it was possible to establish several stages of the NK lytic pathway, the molecular and biochemical nature of each of these steps are not yet known. Several approaches are currently being investigated, making use of variants selected that lack only one step in the overall chain of reactions. The availability of recombinant, purified LT and TNF and the possible availability of recombinant cytolysin or perforin in conjunction with NKCF should enable us to establish the relationship among these various cytotoxins and their role in the mechanism of cell-mediated cytotoxicity. In addition to fundamental research, the studies clearly are significant for their direct application in clinical medicine. It is reasonable to develop approaches that can either enhance susceptibility of tumor cells to lysis by NK cells or NKCF or to consider treatments that can

185 restore functional NK activity where it is defective. Our laboratory is currently examining some of these potential therapeutic applications.

ACKNOWLEDGMENT Supported in part by Grants CA 35791 and CA 37199 awarded by the National Cancer Institute, Department of Health and Human Services, and in part by grants from the Universitywide Task Force on AIDS, the Cancer Research Coordinating Committee, and the Johnson Comprehensive Cancer Center.

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Griffin, J. P., Hercend, T., Beveridge, R., and Schlossman, S. F. (1983).J.lmmunol. 130, 2947. Grimm, E. A., and Bonavida, B. (1977).J.lmmunol. 119, 1041. Hanna, N. (1983). Sum. Synth. Pathol. Res. 2,68. Herberman, R. B. (1983). “NK Cells and Other Natural Effector Cells.” Academic Press, New York. Hercend, T. (1983). Nature (London)301, 158. Hiserodt, J. C., Bretvan, L. J., and Targan, S. R. (1982a).J. lmmunol. 129, 2266. Hiserodt, J., Britvan, L. J., and Targan, S. (1982b).J. lmmunol. 129, 1782. Hoffman, T., Hirata, F., Bougnoux, P., Fraser, B. A., Goldfarb, R. J., Herberman, R. B., and Axelrod, J. (1981). Proc. Natl. Acad. Sci. U.S.A.78,3839. Hudig, D., Havertz, T., Fulcher, C., Redelman, D., and Mendelsohn, J. (1981).J. lmmunol. 126, 1569. Jensen, P. J., and Koren, H. S. (1979).J. Immunol. 123, 1127. Kahle, R., Hiserodt, J., and Bonavida, B. (1983). Cell lmmunol. 80, 97. Katz, P., Zaytoun, A. M., and Fauci, A. S. (1982).J. lmmunol. 129, 187. Kiessling, R., Klein, E., and WigzelI, H. (1975). Eur. J. lmmunol. 5, 112. Kiyohara, T., Lauzon, R., Haliotis, T., and Roder J. C. (1986). In “Immunobiology of Natural Killer Cells” (E. Lotzova and R. B. Herberman, eds.), Vol. 1, p. 108. CRC Press, Boca Raton, Florida. Kondo, L. L., Rosenau, R., and Waren, D. W. (1981).J.lmmunol. 126, 1131. Lanier, L. L., Curla, S., Federspiel, N., and Phillips, J. H. (1986).J.Erp. Med. 163,209. Lavie, G. (1982).In “NK cells and Other Natural Effector Cells” (R. B. Herberman, ed.), p. 939. Academic Press, New York. Mannel, D. N., Moore, R. N., and Mergenhagen, S. E. (1980). Infect. lmmun. 38,523. Matthews, N. (1983). Immunology 48,321. Millard, P. J., Henkart, M. P., Reynolds, C. W., and Henkart, P. A. (1984).J. lmmunol. 132,3197. Moon, T. D., Morlez, J. E., Vesella, R. L., and Lang, P. H. (1983).Scand.J.lmmunol. 18, 255. Neibour, A. P., and’Huberman, H. S. (1982). Eur. J. lmmunol. 128, 123. Newman, W. (1982). Proc. Natl. Acad. Sci. U.S.A.79,3858. Nissen-Meyer, J., and Hemmerstrom, J. (1982). Infect. lmmun. 38, 62. Pennica, D., Nedwin, G. E., Hayflick, F. S., Seeburg, P. H., Palladino, M. A., Kohr, W. J., Oeggarwel, B. B., and Goeddel, D. V. (1984). Nature (London)312,724. Perussia, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G. (1983).J.Immunol. 130,2133. Phillips, W. H., Ortaldo, J. R., and Herberman, R. B. (1980).J. Immunol. 125,2332. Podack, E. R., and Dennert, G. (1983). Nature (London)302,442. Quan, P., Ishizaka, T., and Bloom, B. R. (1982).J. lmmunol. 128, 1786. Roder, J. C., and Klein, E. (1979).J. lmmunol. 123, 2785. Roder, J. C., and Lauzon, R. (1983). Sum. Synth. Pathol. Res. 2, 82. Roder, J. C., Kiessling, R., Biberfeld, P., and Anderson, B. (1978).J.lmmunol. 121,2509. Roder, J. C., Ahrlund, R., and Jondal, M. (1979a).J. E x p . Med. 150, 471. Roder, J. C., Rosen, A., Fenyo, E. M., and Troy, E. A. (1979b). Proc. Natl. Acad. Sci. U S A . 76, 1405. Roder, J. C., Argov, S., Klein, E., Petersson, C., Kiessling, R., Anderson, K., and Hansson, M. (1980). lmmunology 40, 107. Roder, J. C., Karre, K., and Kiessling, R. (1981). Prog. Allergy 28,66. Roozemond, R. C., Urli, D. C., Wright, S. C., Graves, S. S., and Bonavida, B. (1985).J. lmmunol. 134,2209.

187 Roozemond, R. C., van der Geer, P., and Bonavida, B. (1986).J. Immunol. 136,3921. Rosenberg, E. B., McCoy, J. L., Green, S. S., Konelly, F. C., Swarski, D. F., Levine, P. H., and Herbennan, R. B. (1974). J. Natl. Cancer Inst. 52,345. Ruddle, N. H., and Waksman, B. H. (1968). J. E x p . Med. 128, 1267. Schmid, D. S., Powell, M. B., Mahoney, K. A., and Ruddle, N. H. (1985). Cell. Immunol. 93, 68. Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W., and Wallace, R. B. (1985). Nature (London)313,803. Stutman, O., Dien, P., Wisun, R. E., and Lattime, E. C. (1980). Proc. Natl. Acad. Sci. U.S.A.77,2895. Takasugi, M., Mickey, M. R., and Terasaki, P. I. (1973).Cancer Res. 33,2898. Targan, S., Grimm, E. A., and Bonavida, B. (1980). J. Clin. Lab. Immunol. 4, 165. Trinchieri, G., Santoli, D., Dee, R. R., and Knowles, B. B. (1978).J. E x p . Med. 147,1314. Vodinelich, J., Sutherland, R., Schneider, C., Newman, R., and Greaves, M. (1983). Proc. Natl. Acad. Sci. U.S.A.80,835. Walker, S. M., and Lucas, Z. J. (1973). Transplant. Proc. 5, 137. Wang, A. M., Greasy, A. A., Ladner, M. B., Creasey, A. A., Liu, L. S., Strickler, J., Jauelle, N., Arsdell, V., Yamamoto, R., and Mark, D. F. (1985). Science 228, 149. Ware, C. F., and Granger, G. A. (1981).J. Immunol. 126, 1934. Warner, J. F., and Dennert, G. (1982). Nature (London) 300, 31. Weissman, G., Holdstein, F., Hoffstein, S., and Tsung, P. K. (1975).Ann. N.Y. Acad. Sci. 253,1750. Weitzen, M. L., Innis, E., Yamamoto, R. S., and Granger, G. A. (1983a). Cell. lmmunol. 77,42. Weitzen, M. L., Yamamoto, R. S., and Granger, G. A. (198313). Cell lmmunol. 77, 31. Williams, T. M., and Granger, G. A. (1968). Nature (London)219, 1076. Wright, S. C., and Bonavida, B. (1981).J. Immunol. 126, 1516. Wright, S. C., and Bonavida, B. (1982).J. Immunol. 129,433. Wright, S. C., and Bonavida, B. (1983a). Proc. Natl. Acad. Sci. U.S.A. 80, 1688. Wright, S. C., and Bonavida, B. (1983b).J. Immunol. 130, 2965. Wright, S. C., and Bonavida, B. (1984a). In “Natural Killer Activity and Its Regulation” (T. Hoshino et al., eds.), p. 145. Excerpta Medica, Amsterdam. Wright, S. C., and Bonavida, B. (1984b). J. Immunol. 123,3415. Wright, S. C., and Bonavida, B. (1984~).In “Natural Killer Activity and Its Regulation” (T. Hoshino et al., eds.), p. 138. Excerpta Medica, Amsterdam. Wright, S. C., and Bonavida, B. (1987).J. Immunol. 138, 1791. Wright, S. C., and Bonavida, B. (198613). Submitted. Wright, S. C., and Bonavida, B. (1986~).In “Cytotoxic Lymphokines and Cancer: Biological and Chemical Aspects” (J. H. Ranson and J. Ortaldo, eds.). Humana Press, New Jersey. (In press). Wright, S. C., Weitzen, M. L., Kahle, R., Granger, G. A., and Bonavida, B. (1983). J. Immunol. 130,2479. Wright, S. C., Wilbur, S. M. and Bonavida, B. (1985). Nut. lmmun. Cell Growth Regul. 4, 202. Wright, S. C., Kane, K., Clark, W. R., and Bonavida, B. (1986). Submitted. Yanagi, (1985). Nature (London)314, 631. Zascharchuk, C. M., Drysdale, B. E., Mater, M. M., and Shein, H. S. (1983). Proc. Natl. Acad. Sci. U S A . 80, 6341.

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SHEDDING OF HUMAN TUMOR-ASSOCIATED ANTIGENS IN VlTRO AND IN VlVO Meenhard Herlyn, Ulrich Rodeck, and Hilary Koprowski The Wistar Institute of Anatomy and Biology. Philadelphia, Pennsylvania 19104

I. Introduction

The available repertoire of human tumor markers has increased considerably with the recent development of monoclonal antibodies (MAbs). This article focuses on the tumor-associated antigens (TAAs) defined with MAbs that are shed in vitro and in viuo. TAAs are those glycoproteins and glycolipids that are expressed on the surface or in the cytoplasm of tumor cells and more often than not are detected on either the normal counterpart of tumor cells or on fetal cells. The term shedding, as used in this article, implies the release of antigenic components, in soluble or particulate form, from cells into the tissue culture medium or into blood or other human secretions. Antigens can be released by cells as a function of active metabolic turnover or as a result of cell death. Shedding of antigens by normal cells as a physiological phenomenon in vitro and in uiuo has been reviewed by Black (1980). The immunization of mice with human tumor cells or cellular components, followed by fusion of spleen cells with mouse myeloma cells, has yielded MAbs that detect a variety of tumor-associated glycolipid and glycoprotein antigens. In this article, we discuss those MAbs that detect TAAs expressed by human carcinomas of colon, rectum, stomach, breast, pancreas, bladder, prostrate and lung, and melanoma since shedding of antigenic components has been studied mostly in these tumor types. Cultures of these tumors provided the most suitable model for the study of TAA shedding. In this article we shall discuss the following problems: (1)Which type of tumor or cells shed TAAs in uitro? (2) Is there a modulation of the shed antigens compared to the antigens bound to the tumor cells? (3) Are there antigenic and other differences between antigens shed in culture as compared to those detected in blood of cancer patients? (4)Can shed189 ADVANCES IN CANCER RESEARCH, VOL. 49

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction In any form reserved.

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MEENHARD HERLYN ET AL.

ding be influenced by exogenous and endogenous factors? ( 5 ) Which TAAs are shed in vivo? Finally, we summarize the current information on the presence in cancer patients of shed antigens in complex with antibodies. Only a few antigens in immune complexes have been defined with MAbs. The number of MAbs used in serodiagnostic studies is increasing rapidly, and we discuss here the best studied markers. As more clinically relevant tumor markers are defined with MAbs, we might begin to understand the significance of tumor antigen shedding in cancer patients. II. Shedding of Tumor-Associated Antigens in Vitro

A. PATTERN OF ANTIGENSHEDDING IN TISSUE CULTURE AS DEFINED BY MONOCLONAL ANTIBODIES 1. Cellular Specijicity of TAA Shedding in Tissue Culture

Each type of tumor when maintained in tissue culture displays a characteristic pattern of TAA shedding. Table I gives examples of TAAs shed by colorectal, gastric, pancreatic [gastrointestinal (GI) tract], and mammary carcinoma and melanoma cells. The major blood group antigens A, B, H, and related TAAs as well as Lewis and related TAAs are shed predominantly by GI tract carcinoma cells and not, or only rarely, by other carcinoma cells. A common feature of GI tract,

TABLE I MONOCLONAL ANTIBODY-DEFINED TUMOR-ASSOCIATED ANTIGENSSHEDBY CULTURED GASTROINTESTINAL CARCINOMA, MAMMARY CARCINOMA, OR MELANOMA CELLS

GI tract carcinoma _

_

Breast carcinoma _

A, B, and H blood group antigens and related carbohydrate structures Lewis antigens and related structures X and Y antigens and related carbohydrate structures CEA and related proteins 30- to 100-kDa proteins

_

~ ~

~

Melanoma

~

Milk fat globule membrane protein, X and Y antigens and related carbohydrate structures CEA and related proteins 30- to 100-kDa proteins

Basement membrane proteins and related proteins Disialogangliosides 30- to 100-kDa proteins Cytoplasmic proteins

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

191

breast, and ovarian carcinoma cells is shedding of X- and Y antigens. Carcinoembryonic (CEA) antigen and CEA-related proteins of low molecular weight are found in both GI tract and mammary carcinomas. Malignant melanoma and glioma shed large quantities of extracellular matrix proteins, such as fibronectin and related high-molecular-weight proteins, Minor brain disialogangliosides such as GDZ and GD3 are major melanoma-associated antigens. Cells of all tumor types release various low molecular weight proteins.

2. Antigen Specijicity of Shedding Expression and shedding of TAAs follow a characteristic pattern which can be divided into four groups (Table 11). The first group consists of those antigens that are secreted at high levels but are weakly or not expressed on the tumor cell surface. Examples are Lewis and A, B, and H antigens for GI tract carcinoma cells and extracellular matrix proteins for melanoma cells. Common to all solid tumors studied are a variety of shed cytoplasmic antigens. Melanoma cells, for example, shed a 80-kDa protein (Herlyn et al., 1985c) or a dimer of a major 94-kDa and a minor 72-kDa component (Natali et al., 1982). The second group consists of those TAAs that are both shed and expressed by tumor cells. Examples in colorectal carcinoma (CRC) are X antigen and CEA, and in melanoma, disialogangliosides and HLA class I and class I1 antigens. In nine CRC cell lines, Shi et al. (1983) found 80% of CEA associated with the cell membrane and 20% shed into the culture medium. Cell lines with decreased cellular CEA due to higher cell densities shed increased quantities into the culture medium. The third group includes antigens expressed by normal and malignant cells of heterologous cell populations which are shed by one type of cells only. For instance, a 200-/80-kDa melanoma-associated antigen that was found expressed on the surface of various malignant and TABLE I1 PATTERN OF ANTIGEN EXPRESSION AND/OR SHEDDING BY CULTURED TUMOR CELLS Group I Group I1 Group I11 Group IV

Antigens secreted at high levels but either weakly or not expressed on the cell surface Antigens both expressed on cell surface and secreted Antigens expressed by cells of different types but shed only by cells of one type Cell-bound antigens (not shed)

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MEENHARD HERLYN ET AL.

normal cells such as GI tract carcinoma, lymphocytes, or fibroblasts was shed only by cells of neural crest origin, e.g., melanoma and astrocytoma (Herlyn et al., 198313). When MAbs were selected for binding specificity on spent medium of cultured cells (Herlyn et al., 1985b), approximately 25% of MAbs that showed restricted binding on the spent medium cross-reacted with cells of many different histotypes, suggesting that differential expression and shedding of antigens is a relatively common phenomenon. Another subgroup consists of TAAs related to the stages of tumor progression within the same histotype. The 120-kDa antigen of MAb 77.1 is expressed by cells of nevi, primary and metastatic melanoma (Herlyn et al., 1985e) but, as illustrated in Fig. 1, is shed into the FM 56 FM 60 FM 61 FM 70 FM 72 FM 74

Normal Skin

WML 769-3 WML 817 WML 829 WML 837 WML848 ---WM 245 -------WM 39 WM75 WM 98-1 WM 115 WM 278 WML 793 WML 853-2 WM 46 WM 164 WM 165-1 WM 239A WM 2664 WM 373 WML 852 SK MEL 23 0

1

Nevus

---_-_____

2 3 CPM x 10-3

4

RGP

5

FIG.1. Reactivity of antimelanoma MAb ME 77.1, which detects a 120-kDa protein, with supernatants of melanocytes isolated from different stages oftumor progression. All cultures were maintained at approximately the same density for 5 days in serum-free supplemented medium. With the exception of melanocytes from normal skin, all cells expressed the 120-kDa antigen.

193

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

medium only by primary and metastatic melanoma and not by cultures of the other types of pigmented lesions or by melanocytes. Thus, shedding of the TAA in tissue culture depends on the stage of tumor progression of tissue used to initiate the cultures. The fourth group consists of those antigens that remain cell bound and are not shed. These are the receptors for epidermal growth factor (EGF), nerve growth factor (NGF), or platelet-derived growth factor (PDGF) present on tumor cells. Cells expressing unusually high concentrations of a receptor on their surface may shed only a small amount of receptor, as, e.g., in the case of the epidermoid carcinoma cell line A431 which expresses approximately 2 X lo6 EGF receptor molecules per cell and releases small quantities of the truncated receptor into the medium. A 40-kDa GI tract carcinoma TAA reactive with MAb CO 17-1A (Herlyn et al., 1979) is not shed in vitro (Steplewski et al., 1981) or in viuo. Similarly, immunization of mice with human placental membranes has yielded MAbs with high specificity for melanocytic cells. A 120-kDa protein antigen expressed by melanoma cells is not shed (Herlyn et al., 1985e).

3. Kinetics of Antigen Shedding Shedding of TAAs by cultured cells into the medium obtains a maximum after 2-5 days in serum-free medium (Fig. 2). Shedding of basement membrane-related proteins increased considerably immediately

1

2

3

4 Days

5

6

7

FIG.2. Reactivity in indirect solid-phase radioimmunoassay of monoclonal antibodies to serum-free tissue culture supernatant of melanoma cell line WM 9. Supernatant was collected at various days, centrifuged, and tested with anti-HLA-DR(O),anti-GD2/ GD3 (0),and 302-9 (fibronectin-relatedprotein) (A).

194

MEENHARD HERLYN ET AL.

after transferring the cells from a serum-containing to a serum-free medium when cell viability is higher than 98%. Other antigens such as gangliosides, chondroitin sulfate proteoglycans, or HLA-DR antigens reach maximum levels when cell viability decreases due to starvation in serum-depleted medium. Cells initially grown in the presence of fetal bovine serum and then maintained under serum-free conditions shed CEA more freely than during logarithmic growth (Alderman et al., 1985). The continuous culture of human cells in chemically defined medium, which has recently been achieved for cells of several normal and malignant histotypes (see Barnes et at., 1985, for review), will facilitate investigations to determine whether MAb-defined TAAs are shed due to an active metabolic turnover or as a result of cell death. 4. Modulation of TAA Shedding

Compounds that influence growth and/or differentiation of cells in culture may also modulate expression and shedding of TAAs. y-Interferon, which is a potent stimulator of the expression of HLA-DR by cultured melanoma, colorectal, and mammary carcinoma cells (Basham and Merigan, 1983; Houghton et al., 1984a; Schwartz et al., 1985; Capobianchi et al., 1985; Herlyn et al., 1985c), increased the shedding of several melanoma-associated antigens, including proteoglycan and disialogangliosides (Giacomini et d . ,1984; Herlyn et a-Interferon enhanced the expression of high- and lowal., 1985~). molecular-weight CEA-related proteins on mammary carcinoma cells (Greiner et al., 1984,1985). CEA shedding by cultured human gastric, pancreatic, and lung cancer cell lines was markedly enhanced by theophylline and prostaglandin Ez (Yamaguchi and Kawai, 1983). B. HIGH-MOLECULAR-WEIGHT, MUCIN-TYPE STRUCTURES Immunizing mice with carcinoma cells of GI tract, breast, ovary, and lung or with semipurified antigenic preparations from these tumors has yielded a large number of MAbs with restricted, although not histotype-specific, binding patterns to tumor tissues. Biochemical analyses of antigens extracted from tumor cells revealed that MAbs detecting carbohydrate structures bound to both glycolipids and glycoproteins (for reviews, see Hakomori and Kannagi, 1983; Hakomori, 1984, 1985; Feizi, 1985). CO 19-9 was the first example of such an MAb (Koprowski et al., 1979); the carbohydrate determinant detected by this antibody was defined as a monosialoganglioside (Magnani et al., 1981), with specificity for sialylated Lewisa (Magnani et al., 1982).

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

195

In sera of cancer patients and in spent medium of cultured cells CO 19-9 bound to a high-molecular-weight (HMW) mucin of 5 x lo6 (Magnani et al., 1983). Table I11 summarizes the HMW, mucin-type antigens, most of which have a molecular weight above 200,000. In addition to the Lewisa complex (Table IV), the X, Y, H complex (Table V), and the milk fat globule membrane antigens (Table XV), several other as yet TABLE 111 HIGH-MOLECULAR-WEIGHT, MUCIN-TYPE ANTIGENSIN SPENT TUMOR CELLSAS DETECTED WITH MEDIUMOF CULTURED MONOCLONAL ANTIBODIES= Antigen MAbs detecting carbohydrate determinants Lewis" and related structuresb X and related structuresb

Milk fat globule membraneb TAG 72b Du-Pan-2 antigenb MAbs detecting protein antigens CEA and related proteinsb

Proteoglycanb MAbs detecting undefined determinantsc Pancreatic cancer-associated antigen Pancreatic oncofetal antigen OC 125 antigenb 1D3 antigen

MOv2 antigen Lung adenocarcinoma antigen YH206

Predominant tumor type

Pancreatic, gastric, and colorectal carcinoma Colorectal, mammary, ovarian, and lung carcinoma Mammary carcinoma Mammary and colorectal carcinoma Pancreatic carcinoma

Colorectal, mammary, and other carcinoma Melanoma

Pancreatic carcinoma Pancreatic carcinoma Ovarian carcinoma Ovarian and colorectal carcinoma Ovarian carcinoma Lung and GI tract carcinoma

See text and following tables for references. Assays for detection of antigens in sera of patients available. c Probably carbohydrate determinants. 0

196

MEENHARD HERLYN ET AL.

unidentified HMW antigens have been detected on pancreatic, ovarian, and lung carcinoma.

1. Gastrointestinal Cancer Antigen (GICA) GICA is a HMW mucin that carries several blood group and related determinants (Table IV). The tumor-associated sialylated Lewisa has been defined with at least five MAbs (CO 19-9, Koprowski et al., 1979; CO 29.11, Herlyn et al., 1985d; CSLEAl, Chia et al., 1985; CA 50, Holmgren et al., 1984; and 121 SLE, E. Herrero-Zabaleta et at., personal communication). CO 19-9 is the most widely used MAb for experimental and clinical studies (see Herlyn and Koprowski, 1985, for review). With an association constant of 3 x lo7M-I, CO 19-9 has a lower binding affinity than CO 29.11 (Herlyn et al., 1985a), but its binding to sialylated Lewisa is more specific since CO 29.11 crossreacts weakly with the nonsialylated Lewisa. Binding of CO 19-9 to target cells is completely inhibited by CO 29.11, whereas binding of CO 29.11 to target is only partially blocked, Multiple antigenic specificities of mucins have been delineated using double-determinant immunoassays (DDIAs) in which the first antibody is coupled to a solid phase and the second antibody is labeled

TABLE IV CARBOHYDRATE DETERMINANTS ON GICA IN SPENTMEDIUM OF CULTURED GASTROINTESTINAL TRACT CARCINOMA CELLS References Determinant

MAb

Production ~~

Sialylated Lewis" Lewis"

co 19-9

Sialylated Lewis" and Lewis" Lewis" and Lewisb

CO 29.11

co 51.2

Lewisb and H type 1

co 10

CO 51.4

Mucin association ~

Koprowski et al. (1979) Blaszczyk et al. (1984cJ98S) Herlyn et al. (198Sb)

Magnani et al. (1982) Herlyn et al. (1985a)

Blaszczyk et al. (1984aJ985) Brockhaus et al. (1981), Blaszczyk et al. (1984a)

Herlyn et al. (198413)

Variably (depending on cell type) present on GICA: oc 12s Bast et al. (1981) Undefined A types 1 and 2

33/25/1/17

Ernst et al. (198413)

B types 1 and 2

PA 83-52

Hansson et al. (1983)

Herlyn et al. (198Sb)

Herlyn et 01. (198413)

R. C. Bast (personal communication) M. Herlyn et al. (unpublished) Herlyn et al. (1984b)

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

197

6-

421

1:4

1

I

1

I

I

I

I

I

196 1:64 1:2% 1:1024 Dilution of spent media of tumor cells

I

I

1:-

FIG.3. Double-determinant immunoassay for the detection of antigen in spent meCO 51.4 (A),or CO 10 (0)were dium. Ascitic fluids containing MAbs CO 19-9,).( absorbed to polystyrene beads, and spent medium of pancreatic carcinoma cell line Capan 2 (solid lines) or melanoma cell line WM 46 (dashed lines) was added. Antigen was traced with 1251-labeledMAb CO 19-9 (input 100,000 cpm/we11/200 pl). Data are mean cpm of duplicate determinations with less than 7% deviation from the mean after subtraction of buffer controls. Arrows indicate the dilution of antigen used equaling 100 units.

with an isotope or enzyme (Fig. 3). Using DDIAs, we have detected several other determinants on the GICA mucin: Lewisa, a determinant common to both Lewisa and Lewisb, and a determinant present on Lewisb and H type 1(Table IV). Depending on the blood group of the original donor, blood group antigens A and B may also be associated with GICA on cells, in spent medium, or in patients’ sera.

2. X , Y, H Complex As shown in Table V, there are at least seven epitopes on a recently defined mucin that carries determinants related to X, Y, and H antigens (Rodeck et al., 198713). Different tumor cells of GI tract, breast, lung, or ovarian carcinoma may express in vitro and in vivo either X, Y, or H or any combination thereof and related antigens, leading to considerable antigenic heterogeneity of the shed mucin. Recently, the production of MAbs that detect sialylated X has been described (Fukushima et al., 1984; Chia et al., 1985; Hirota et al., 1985; Kawahara et

198

MEENHARD HERLYN ET AL.

TABLE V CARBOHYDRATE DETERMINANTS ON A HICH-MOLECULAR-WEIGHT STRUCTURE (X, Y, H COMPLEX) SECRETED BY CULTURED COLOFECTAL, MAMMARY, AND OVARIAN CARCINOMA CELLS Determinant

MAb

Isotype

IgM

X X X related Y Y YB H type 2

GA 29-1 CO 56-22 BR 401 101 BR 15-6A BR 55-2 102

IgM IgM IgG, IgG,, IgG3 IgM

H related Lewisb and H type 1

PRD8

IgM

co 10

IgM ~~

0

References"

~

Brockhaus et al. (1982) Hansson et al. (1983) M. Herlyn et al. (unpublished) Fredman et al. (1983) Rodeck et al. (1987b) Blaszczyk-Thurin et al. (1987) Fredman et 01. (1983), Richert et a1. (1983) Lindgren et al. (1985) Brockhaus et al. (1981), Blaszczyk et al. (1984a) ~

References are to the initial characterization of MAbs.

al., 1985). Most likely, this determinant is shed as an integral part of the X, Y, and H complex. 3. Carcinoernbryonic Antigen (CEA) CEA is one of the best studied tumor markers. Since 1980, several groups have produced MAbs binding to CEA and related proteins in efforts to define the epitopes on this multideterminant structure which is shed by cultured cells (Table VI). Epitopes on CEA can be divided into seven groups, according to the size and nature of the protein(s) detected by MAbs. Group I epitopes are only on one HMW protein. This epitope(s) appears of highest specificity. Group I1 epitopes are present on two HMW proteins. As indicated for Groups 111 and V, HMW CEA share determinants with low-molecular-weight (LMW) proteins, of which NCA (Kleist et al., 1972) has been most extensively studied. Despite the high level glycosylation of CEA, few carbohydrates have been defined on CEA (Group VII). Examples are MAb FH6, which defines a difucoganglioside (Fukushi et al., 1985),and MAb BR 401, which binds to an X-related structure (Table V). Like the carbohydrate structures on glycolipids and on glycoproteins, those on CEA appear to differ when the structure is cell bound as compared to shed CEA circulating in sera. Martin and Halpern (1984) found dramatically different kinetics of clearance of CEA isolated from patients' sera as compared to CEA extracted from tumors grown in nude mice.

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

199

TABLE VI DETECTION OF CEA AND RELATEDANTIGENSWITH MONOCLONAL ANTIBODIES MAb group

Characteristics

Example

Reference

I

Binds one HMW protein (200 kDa)

3d635 Col 1 AS001

Mitchell (1980) Muraro et al. (1985) Yachi et al. (1984)

I1

Binds two HMW proteins (180 and 200 kDa)

35 #4 1 YK013

Accolla et al. (1980) Lindgren et al. (1982) Imai e t al. (1984)

111

Binds two HMW and one LMW (50 kDa)

B1.1 5E91E9 202

Colcher et al. (1981) Kupchik et al. (1981) Buchegger et al. (1982a,b)

IV

Binds one LMW protein (50 or 90 kDa)

B6.2

Colcher et al. (1981)

V

Binds two HMW and two LMW proteins

CO 2032 T84 1

Blaszczyk et al. (1984b) Neumaier et al. (1985)

VI

Binds more than four proteins

B9F10

Herlyn et al. (1983a)

VII

Binds carbohydrate determinants

A21.91 FH6 BR 401

Haggarty et al. (1986) Nichols et al. (1985) M. Herlyn et al. (unpublished)

4 . Other Antigens

The pancreatic cancer-associated antigen (PCAA) (Gelder et al., 1978; Shimano et al., 1981; Loor et al., 1984), as listed in Table 111, has also been detected with MAbs (Shimano et al., 1985). Similarly, three MAbs have been produced by Matsuura et al. (1985) to the pancreatic oncofetal antigen (POA) originally described by Hobbs et al. (1980). These newly available MAbs may increase the specificity of detecting PCAA and POA. The recently characterized Du-Pan-2 antigen (Metzgar et al., 1982, 1984; Lan et al., 1985), a new pancreatic cancer-associated mucin, appears to have a more restricted specificity for pancreas carcinoma cells than other pancreas-associated mucins, e.g., GICA. MAbs to ovarian carcinoma have defined three HMW structures. OC 125 antigen is expressed on more than 80% of epithelial ovarian tumors (Bast et al., 1981) and is shed at high levels both in uitro (Masuho et al., 1984) and in uiuo (Bast et al., 1984; Klug et al., 1984). Detailed biochemical analysis of this structure has recently been published (Davis et d., 1986). The antigens MOv (Miotti et al., 1985;

200

MEENHARD HEEUYN ET AL.

Tagliabue et ul., 1985) and 1D3 (Gangopadhyay et al., 1985) are two recently described mucins with specificities for ovarian carcinoma. To date, only one preliminary report exists on a lung cancer-associated HMW mucin-type antigen detected with MAbs (Yachi et al., 1985). The HMW mucin detected with MAb B72.3 is a 290- to 400kDa protein expressed by mammary carcinoma but also expressed and shed by CRC cells (Colcher et al., 1981; Schlom et al., 1985; Johnson et al., 1986). The chondroitin sulfate proteoglycan is a HMW protein of neural crest tumors. This proteoglycan of >260 kDa, as originally characterized by Bumol and Reisfeld (1982) on melanoma cells, is shed by melanoma and astrocytoma cells in uitro and in uiuo (Ross et al., 1984). At least five determinants have been identified on the proteoglycan molecule (Rettig et d , 1986). Presumably all anti-proteoglycan MAbs bind to peptide moieties and not to carbohydrates. C. PROTEIN ANTIGENS Tumor cells in vitro shed a variety of protein antigens with molecular weights between 20,000 and 150,000. Despite numerous efforts by our laboratory and by others using a large panel of MAbs, “small” proteins (20-200 kDa) have not proven useful as serodiagnostic tumor markers, with a few exceptions such as a-fetoprotein, placental alkaline phosphatase, and human chorionic gonadotropin. As shown in Table VII, seven MAbs to colorectal carcinoma, mammary carcinoma, or melanoma detect antigens that are shed in uitro but not in uiuo.

TABLE VII PROTEIN TAAs SHEDIN CULTURE BUT NOTDETECTED IN PATIENTS’ SERA Cell type used to derive MAb

MAb

Antigen

Reference

Colorectal carcinoma

CO 1472 CO 44.1 GA 9-1

40 kDa N.I.“ 25 kDa

Herlyn et al. (1982) Herlyn et al. (1985a) M. Herlyn et al. (unpublished)

Mammary carcinoma

BR 8-3 BR 66-2

200 kDa 38140142 44 kDa

M. Herlyn et al. (unpublished) Steplewski et al. (1985)

Melanoma

ME DA3

1051130 kDa 120 kDa

Herlyn et al. (1985e)

M E 77.1 Not identified, most likely protein.

Herlyn et al. (1985e)

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

201

Interestingly, most of these antigens belong to a group of antigens that are expressed in vitro by cells of different types but shed only by cells of one type (see Group 111, Table 11). It is unclear whether the absence of these antigens in vivo is due to (1)lack of shedding, (2) rapid degradation, (3)rapid excretion, or (4)insufficient sensitivity of current detection assays. The biophysical property of the shed material may also be important. For example, the 120-kDa melanoma-associated protein detected with ME 77.1 (see Fig. 1)is shed as a considerably larger structure since high-speed centrifugation of spent medium of melanoma cells (100,000 g for 90 min) reduces the binding of MAb to antigen in spent medium by 72%. It is conceivable that such membrane “fragments” are rapidly cleared from the peripheral blood by the reticuloendothelial system. Protein antigens secreted in vitro as well as in vivo include HLA class I and class I1 antigens (Herlyn et al., 1984a), melanoma-associated p97, a highly glycosylated melanoma antigen of 20-50 kDa (Atkinson et al., 1985; Donoso et al., 1985) (Table XVI), and the LMW CEA-related proteins (Table VI). D. GLYCOLIPID ANTIGENS Carbohydrate structures, as discussed in Section II,B, may also be shed as glycolipids. The biochemical nature of shed glycolipid antigens is poorly defined. These antigens may rapidly adhere to each other, forming micelles, or, as in the case of Lewis glycolipids, adhere to circulating red blood cells. For the study of shed glycolipid antigens, spent media and sera of patients may be extracted with chloroform :methanol as described by Schulz et al. (1984) and Thurin et al. (1985). Melanoma-associated gangliosides that are secreted at high levels in vitro are not associated with proteins. The only possible exception is MAb M E 50.5 which not only binds to GD3 but also to a protein (M. Thurin, personal communication). MAbs to disialogangliosides are currently being used in immunotherapy trials in patients with melanoma (Houghton et al., 1984b; D. Guerry et al., unpublished), owing to their high binding specificity and potent growth suppression of human tumors in nude mice. 111. Shedding of Tumor-Associated Antigens in Vivo

A. GENERAL CONSIDERATIONS Several distinct applications for the immunodiagnosis of human tumors might be considered using antibodies to detect TAAs in patients’

202

MEENHARD HERLYN ET AL.

sera. First, immunodiagnostic approaches might be useful in the early detection of cancer through screening high-risk population groups. Second, such procedures may aid in distinguishing between patients with malignant and those with benign disease. Third, immunoassays might have prognostic value, to direct therapy modalities, to monitor the patients' response to therapy, and to detect possible early recurrences. TAAs, as studied to date, do not display a complete specificity for a given tumor cell type. Antigens may be expressed in different quantities on normal cells and their malignant counterparts, or they may be absent on normal cells of the same histotype as the tumor but present on other normal cells. The ultrastructural location of a given antigen may differ widely in normal and malignant cells. Such differences, e.g., antigen expression on the tumor cell surface versus its cytoplasmic presence in normal cells, can be exploited for immunodiagnostic purposes. Antigens on the HMW, mucin-type human milk fat globule (HMFG) membrane are immunohistological markers for breast carcinoma despite their presence in the ductal epithelium of the normal lactating breast. As shown in Table VIII, HMFG membrane proteins are localized differently in normal and malignant tissues: Staining with antiHMFG MAbs, for example, is localized in the luminal surface of nor-

TABLE VIII In Situ LOCALIZATION OF HIGH-MOLECULAR-WEIGHT BREAST CARCINOMA-ASSOCIATED ANTIGENSFOUND AT ELEVATED LEVELSIN SERAOF CANCER PATIENTS Localization of antigen" MAb

Antigen

Normal tissue

Malignant tissue

HMFG 2 DF3 F36122

280/230 kDa 330/450 kDa HMW glycoprotein N.I.b >400 kDa

Extracellular Apical Apical/luminal

Cytoplasmic Cytoplasmic Cytoplasmic

Luminal Apical

Cytoplasmic Cell membrane/ cytoplasmic

Not done

Cytoplasmic"

3E 1-2 115D8 B72.3

220-400 kDa

As tested in immunoperoxidase assays. Not identified. c Luminal binding on well-differentiated tumor sections. 0

b

Reference Berry et al. (1985) Kufe et al. (1984) Papsidero et al. (1983a) Stacker et al. (1985) Hilkens et al. (1984) Horan Hand et al. (1983)

203

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

ma1 tissues but is cytoplasmic in malignant tissues. Differences in cellular localization of the antigens may also account for differences in shedding of an antigen either into the lumen of a glandular tissue type or, after transformation of cells, into the circulation.

B. METHODOLOGICAL APPROACHESTO DETECTION Four basic approaches are currently available for the detection of antigen in patients’ sera (Table IX). The DDIA is highly sensitive, reproducible, easy, and inexpensive to perform. MAb is adsorbed to a solid phase such as polystyrene beads, incubated with serum, and a labeled second antibody is added to “trace” the antigen. The same antibody can be used as both “catcher” and “tracer” if the antigenic determinant detected by the MAb is repeatedly present on the molecule, e.g., carbohydrates on HMW, mucin-type structures. For CEA, proteoglycan, and other proteins, the second MAb should bind to a different antigenic determinant. The antibody inhibition assay, which is generally used in early screening studies, does not require purified antibody or antigen. However, this assay is suitable only for antibodies with high binding afhities and requires an optimal target preparation, involving costly cell extraction procedures for each antibody. Idiotype-antiidiotype and antigen inhibition assays are not commonly used. Figure 3 illustrates a DDIA using spent medium of a pancreatic

TABLE IX DETECTION OF ANTIGENSIN PATIENTS’ SERAWITH MONOCLONAL ANTIBODIES Approach

Principle of assay

Reference

Double-determinant immunoassay (DDIA) (sandwich assay) Antibody inhibition assay

One MAb Two MAbs Three MAbs Direct assay (labeled MAb) Indirect assay

Herlyn et al. (1982)

Idiotype-antiidiotype assay

Inhibition of binding of (labeled) antiidiotype to idiotype antibody

Patocnjak et al. (1982), Thomson et al. (1985)

Antigen inhibition assay

Inhibition of binding of labeled antigen to antibody

Chang et al. (1980)

DelVillano et al. (1983) Herlyn et al. (1983a) Buchegger et al. (1982)

-

204

MEENHARD HERLYN ET AL.

carcinoma and a melanoma cell line as positive and negative controls, respectively. A standard curve is used to convert the bound cpm into units/ml, choosing arbitrarily a dilution of the positive standard as 100 units. This unit system was necessary in the development of assays for most HMW, mucin-like structures, except CEA, since purified antigen was not available for quantitative evaluation of results. By testing a large number of sera from healthy donors and from patients with benign and malignant diseases in DDIA, a “cutoff’ level is chosen at which no or a low percentage of control sera are positive (Fig. 4). Despite the arbitrary aspects of the system, the DDIA results of different laboratories have been remarkably similar.

FIG.4. Double-determinant immunoassay using anti-GICA MAb 29.11 both as catcher and tracer of antigen in sera of patients with colorectal carcinoma (A), nonmalignant gastrointestinal diseases (B), and of healthy donors (C). The cutoff between positive and negative sera was at 80 units. Units were calculated using spent medium of pancreatic carcinoma cell line Capan 2 as positive control. Melanoma cell line WM 46 served as negative control.

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

205

The sensitivity of current immunological assays remains relatively low. For a radioimmunoassay or an enzyme-linked assay, approximately lo7 to lo8 binding sitedm1 of serum are necessary for detection. Detection of smaller quantities of circulating antigen awaits considerable improvement on currently available immunoassays. C. PRESENCE OF BLOOD GROUPAND BLOODGROUP-RELATED ANTIGENSIN SALIVA The Lewis antigens are found in various secretions, including those from gastric mucosa and small intestine, and in body fluids such as milk, urine, semen, and saliva (see Hakomori, 1985, for review). Lewis antigens and other carbohydrate antigens such as the X antigen are present as glycolipids and glycoproteins (Pak et al., 1984) in saliva. The Lewis phenotype of individuals can be determined by testing binding of anti-Lewis MAbs to saliva (Steplewski et aZ., 1983). Brockhaus et al. (1985) detected sialylated Lewisa (GICA) in saliva of Lewis-positive individuals, where it is associated with a HMW glycoprotein (Pak et al., 1984). The results of testing salivas of the same individuals for the presence of Lewisa, Lewisb, and GICA are shown in Table X. Of 152 randomly selected salivas, 63% were positive for Lewisa, 62%for Lewisb, and 69%for GICA. GICA secretion is highest in individuals who are high Lewisa secretors, i.e., GICA levels are low or negative in low Lewisa secretors and in high Lewisb secretors. No GICA is detected in Lewis-negative individuals. These quantitative differences in GICA and Lewis antigen expression probably reflect competition of glycosyltransferases for precursor chains that are available for conversion to the sialylated Lewisa antigen. The level of conversion is higher in Lewisb than in Lewisa individuals (Brockhaus et al., 1985). Lewis-negative individuals, as tested in saliva, have no detectable GICA (or Lewis) in normal or malignant GI tract tissues or in their sera. Because high Lewisa secretors may have a higher “background’’ level of GICA in sera than Lewisb individuals, the DDIA cutoff level of GICA in sera is adjusted higher in Lewisa- (80 units) than in Lewisb- (24 units) positive sera (Herlyn et al., 1985d). D. CIRCULATING TUMOR-ASSOCIATED ANTIGENSIN CANCER PATIENTS Since 1981, MAbs have been used in serodiagnostic studies for the detection of circulating TAAs (Koprowski et al., 1981). Table XI summarizes the best studied tumor markers for solid human tumors. The commercial availability of some markers (sialylated Lewisa, CEA,

206

MEENHARD HERLYN ET AL.

TABLE X DETECTION OF GICA AND LEWISDETERMINANTS IN SALIVA^ Salivas reactive with anti-Lewis MAbsb Same salivas binding CO 19-9 (anti-GICA) (cpm)

MAb

CPm

Samples reactive of 152 tested (%)

51.4 (Lewis“)

<500 (negative)

26 (17)

13

10

3

0

500-2000 2001-4000 >4000 Total positive

57 (37) 13 ( 8 ) 26 (17) 96 (63)

17 4 1

36 7 3

4 2 18

0 0 4

57 (37)

28

12

13

4

11 (7) 24 (16) 60 (39) 95 (62)

3 6 21

6 17 32

2 1 7

0 0 0

30.1 <500 (negative) (Lewisb) 500-2000 2001-4000 >4000 Total positive

<500

500-2000

2001-4000

>4000

a Salivas of donors with various benign and malignant disease and of normal donors were tested at a dilution of 1:80 for binding of MAbs in indirect radioimmunoassay with ’~5I-labeledgoat Ig anti-mouse F(ab’)*at 40,000 cpm/well/50 pl. Tissue culture supernatants of MAbs were diluted 1: 10. cpm obtained with supernatants of myeloma were subtracted. All samples were tested in duplicate with less than 7% deviation from the mean.

HMFG membrane-associated glycoprotein) has allowed large-scale studies on patients’ sera to determine the usefulness and limitations of each marker. Initial studies have focused on the feasibility of an assay, e.g., percentage of positive sera in patients with advanced cancer (“true positives”) versus percentage of positive sera in healthy donors TABLE XI WELL-STLJDIED TUMOR MARKERS FOR SERODIAGNOSTIC STUDIES USING MAbs Disease

Antigen

Colorectal carcinoma Breast carcinoma Ovarian carcinoma Pancreatic carcinoma Testicular tumors

Sialylated Lewis’, sialylated X, and CEA Milk fat globule membrane protein OC 125 antigen Sialylated Lewis” and Du-Pan-2 antigen Placental alkaline phosphatase

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

207

and patients with benign diseases of the same tissue type (“false positives”). Patients with minimal disease were also of interest to determine the sensitivity of an assay for early cancer detection. With few exceptions, e.g., CEA, sialylated Lewisa, and OC 125 antigen, most markers have not been tested extensively for their presence in sera of patients with unrelated benign and malignant diseases. 1 . Colorectal Carcinoma a. GZCA. In initial studies for GICA using antibody inhibition assays and MAb 1116-NS-52a (Koprowski et al., 1981; Herlyn et al., 1982), 65% of patients with advanced colorectal carcinoma were found to have elevated levels of GICA compared to 10% of patients with early disease (Dukes A and B). For patients with benign gastrointestinal diseases such as polyposis or inflammatory disease, only 2.5 or 3.8%, respectively, of sera were positive. Healthy donors had a mean false positive rate of 2.2%. Similar results were obtained by others (DelVillano et al., 1983; Ritts et al., 1984; Sears et al., 1982, 1985). Currently, GICA is widely used as a marker for serodiagnostic studies in colorectal cancer (Yoshikawa et al., 1985; Gupta et al., 1985; Staab et al., 1985). Table XI1 summarizes our results on the DDIAs of sera from patients with early and advanced colorectal carcinoma, benign gastrointestinal diseases, and from individuals without evidence of disease (Herlyn et al., 1984b, 1985b,d). Using an MAb to the blood group determinant Lewisb and H type 1(CO 10) as catcher of GICA in sera, followed by tracing GICA with 1251-labeledCO 19-9, 64% of sera of advanced colorectal carcinoma patients were positive versus 2% “false-positive” among control sera (Table XII). Similar results were obtained using an MAb to Lewisa (CO 51.4) or an MAb to sialylated Lewisa and Lewisa (CO 29.11) as antigen catcher. Each combination, however, requires the use of a different cutoff between positive and negative sera, e.g., 35 unitdm1 for CO 19-9/CO 19-9, 24 unitdm1 for CO lO/CO 19-9, and 80 unitdm1 for CO 51.4/CO 19-9. Despite the fact that different sera were tested with different combinations of MAbs at different times, it became clear that anti-Lewis MAbs as catchers increased the sensitivity of detecting GICA. In a direct comparison (Table XIII), we showed that the combined positive results of DDIAs using CO 19-9 and CO 10 as catcher were considerably higher for sera of patients with early malignant disease than by either assay alone. This increased sensitivity of GICA detection in sera of patients with early malignancy resulted in only a marginal increase of false-positives.

TABLE XI1 DETECTION OF CICA IN SERAOF PATIENTS WITH GASTROINTESTINAL DISEASE Percentage of sera positive in DDIA with catcher/tracer MAba

CO 19-9/CO 19-9' Disease Colorectal carcinoma Advanced Early Benign gastrointestinal diseases Polyposis Inflammatory diseases No disease

CO 51.4/CO 19-9

CO 29.11/CO29.11

CO 29.111CO 19-9

Number tested

Positive

Number tested

Positive

Number tested

Number tested

Positive

Number tested

326 120

65 10

76 120

64

49 81

69 13

100

75

60 18

55 n.t.c

60

20

126 159

2.5 3.8

87 139

4 5

67 92

2 3

97 132

5 2

66 102

1 5

240

2

133

4

151

1

78

2

%

2.2

469 ~~~

a

CO 1O/CO 19-9' %

Positive

%

~

DDIAs were performed with sera from different individuals at different times. Combined results of several studies. Not tested.

%

%

Positive

-

209

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS TABLE XI11 INCREASED SENSITIVITY OF DETECTING GICA IN SERAOF PATIENTS WITH COLORECTAL CARCINOMA (CRC) BY COMBINING RESULTSFROM DDIAs Percentage of sera positive using CO 19-9 as tracer

Disease

Number of sera tested

Catcher CO 19-9

Catcher CO 10"

Combined positive results

69 44 28 172

68 15 17 3

63 24 29 4

67 34 42 5

240

2

1

3

Advanced CRC Early CRC Early recurrence CRC Nonmalignant gastrointestinal diseases Healthy donors ~~

Detecting Lewisb and H type 1.

b. Sialylated X Antigen. MAb CSLEXl detected elevated levels of sialylated X antigen in 26%(Hirota et al., 1985)and 25% (Kawahara et al., 1985) of sera with colorectal carcinoma. Despite the low percentage of positive sera, sialylated X antigen may become an important additional (to GICA and CEA) marker in serodiagnostic studies of GI tract carcinomas. c. CEA. Several studies, including those listed in Table VI, have compared the results of MAbs for CEA detection with conventional assays, and, in general, the results are in considerable agreement TABLE XIV DETECTION OF CEA AND RELATEDANTIGENS IN SERA OF PATIENTS USINGMAb 2032 (GROUP V) AS TRACER OF CIRCULATING ANTIGEN ~~~~

~

~~

~

~

~

~

~

~~

Percentage of positive sera with catcher MAb Disease

Number of sera tested

3D6-35 (Group I)"

#4 1 (Group 11)

B6.2 (Group IV)

51 19 47

57 3 4

66 3 2

66 21 18

106

0

0

7

Colorectal carcinoma Advanced Early Nonmalignant gastrointestinal diseases No disease _ 0

_

_

_

~

~

See Table VI for group designation.

2 10

MEENHARD HERLYN ET AL.

(Buchegger et al., 1982a,b). Some anti-CEA MAbs appear to detect CEA with higher specificity than conventional assays (Herlyn et al., 1983a). Table XIV shows that MAbs of Groups I and 11, detecting either one or two HMW proteins, but not MAbs of Groups I11 or IV (see Table VI for group designations) have a low false-positive rate in sera of healthy individuals and of patients with nonmalignant gastrointestinal diseases (Herlyn et al., 1983a). These results were recently confirmed by Lin et al. (1985). d. TAG 72. MAb B72.3 detects a mucin (TAG 72) in sera of patients with colorectal carcinoma with a 56% positivity rate (D. Colcher, personal communication). This antigen was not elevated in sera of patients with nonmalignant GI tract diseases nor in sera of healthy donors. 2. Pancreatic Carcinoma GICA is to date the best serodiagnostic tumor marker for pancreatic carcinoma. It is elevated in more than 80% of sera of cancer patients (Koprowski et al., 1981; Herlyn et al., 1982). Despite its presence in normal pancreatic secretions, GICA is elevated in only 6-8% of patients with nonmalignant pancreatic diseases (Ritts et al., 1984; Schmiegel et al., 1985). Du-Pan-2 antigen was elevated in 68% of sera from patients with pancreatic carcinoma (Metzgar et al., 1984). In contrast to GICA, only 10% of colorectal carcinoma patients had elevated Du-Pan-2 antigen levels.

3 . Mammary Carcinoma In recent years, over 20 MAbs reacting with cell surface determinants or secretory products of mammary epithelium have been studied in detail (reviewed in Schlom et al., 1985). Six different carbohydrate determinants were identified as epitopes on highmolecular-weight glycoproteins in patients’ sera (Table XV). Five of these structures show a distinct pattern of distribution on benign and malignant breast tissue. The clinical relevance of most of these MAbs in early diagnosis and management of breast cancer patients has yet to be proved. Serum levels of HMFGl do not seem to relate to prognosis, whereas high serum levels of HMFG2 correlate with early death of tumor patients (Burchell et al., 1984). In initial studies, F36/22 and Cal showed high rates of false-positive values in benign breast disease (Papsidero et al., 1984; Goodall et al., 1985). A detailed study of 115D8 indicates that antigen serum levels correlate with tumor stage (Hilkens et al., 1986);

TABLE XV HIGH-MOLECULAR-WEIGHTGLYCOPROTE~N ANTIGENSDETECTED IN SERA OF BREAST CANCER PATIENTS ~

_

_

_

_

~

% Positive sera from

MAb

Antigen

HMFG 1 HMFG 2

2801320 kDa 280/320 kDa

DF3

330/320 kDa

F36122

HMW glycoprotein

Cal 115D8

340/390 kDa >400 kDa

a

Human milk fat globule.

Immunizing agent

Healthy controls

Patients with benign disease

HMFG" HMFG (from milk) Membranes of breast cancer cells Breast cancer cell lines

6 16.6

-

-

30 53

Burchell et al. (1984) Burchell et al. (1984)

5

0

72

Hayes

3

13

50

Papsidero et 01. (1984)

0 2.2

13 3

50 43.7

Goodall et al. (1985) Hilkens et al. (1986)

-

HMFG

Patients with malignant disease

Reference

et

al. (1985)

212

MEENHARD HERLYN ET AL.

the highest percentage of positive sera (79%) as well as the highest antigen levels were found in stage IV advanced disease, whereas only 24% and 21% of patients in Stage I and 11, respectively, had elevated serum levels. Sequential determinations of 115D8 levels to monitor the course of disease during therapy appear to be promising. Similar results were obtained in another study using a commerically available DDIA consisting of MAbs 115D8 and DF3 (Rodeck et al., 1987a). Enzyme immunoassay and DDIA using DF3 as catcher and tracer revealed a comparable percentage of positive sera (72 and 76 percent, respectively) in metastatic breast cancer (Hayes et al., 1985). 4 . Ovarian Carcinoma

MAbs to HMW, mucin-type antigens in ovarian carcinoma have been developed (see Table 111).OC 125 antigen is elevated in 82% of epithelial ovarian carcinoma (Bast et al., 1983; Klug et al., 1984), and the level of this antigen correlates with progression or regression of disease in 93% of cases. For benign diseases, a 6% false-positive rate was reported (Klug et al., 1984). Combination assays of OC 125 antigen and alkaline phosphatase may improve detection sensitivity (Eerdekens et al., 1985), whereas GICA and CEA detection are superior to measurement of OC 125 antigen alone in monitoring patients with epithelial ovarian carcinoma (Bast et al., 1984). In a recent study (Miotti et al., 1985), MOv2 antigen was found to be elevated in 8 of 10 effusions from patients with well-differentiated ovarian tumors and in 5 of 11 effusions from patients with poorly differentiated ovarian tumors.

5. Prostate, Bladder, and Lung Carcinoma Wang et al. (1979) described a small glycoprotein [prostate antigen (PA); 34 kDa] localized in normal and malignant prostatic epithelial tissue. This antigen was shown to be released by tumor cells in vitro and in uiuo (Papsidero et al., 1981). Its presence in sera of prostate cancer patients could be demonstrated in immunoassays using heterologous antisera (Papsidero et al., 1980; Kuriyama et al., 1982; Killian et al., 1985). Circulating PA was reported to be of similar molecular weight as tissue PA (Papsidero et al., 1980). Prostate antigen thus represents an exception to the general observation that only HMW glycoproteins (>150,000 MW) can be detected circulating in cancer patients. Whereas most of the other shed TAAs lack tissue specificity, PA expression on human tissues seems to be restricted to prostate epithelium (Wang et al., 1982). Although the development of MAbs to different epitopes of PA has been descirbed (Frankel et al., 1982;

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

2 13

Papsidero et al., 1983b),all serum assays reported so far rely on heterologous antisera. High PA levels in prostatic cancer patients are correlated with poor prognosis and, in postoperative monitoring, strongly suggest disease recurrence (Killian et al., 1985). A number of MAbs reacting with antigens present on urinary bladder carcinoma cells have been reported. The antigens described are predominantly glycoproteins ranging from 17 to 200 kDa. Recent studies show that bladder carcinoma cells express “Ca” antigen (Czerniak and KOSS, 1985) and have an altered expression of blood grouprelated antigens (Coon et aZ., 1985). It remains to be determined whether these or other bladder carcinoma-associated antigens are shed antigens. Few markers have been developed for the detection of lung carcinomas. Sialylated X antigen is reportedly elevated in 65% of sera from patients with lung cancer (Hirota et al., 1985). 6. Melanoma and Other Tumors

Table XVI summarizes the serodiagnostic studies in melanoma patients. For all studies, only patients with advanced disease showed elevated levels of melanoma-associated antigens. None of the assays have been widely applied for monitoring sera in established disease. In neuroblastoma, 21 out of 23 sera of patients had elevated levels of the disialoganglioside GD2 (Schulz et al., 1984).This marker may also prove useful for serodiagnosis of other tumors of neural crest origin such as melanomas. TABLE XVI DETECTION OF ANTIGEN CIRCULATING IN SERAOF PATIENTS WITH ADVANCED METASTATICMELANOMAUSINGMAbsO ~ _ _ _ _

Antigen -~

MAbs

Proteoglycan

p20-50 $00 0

~

Number of sera tested

% Positive sera

66

16

Herlyn et al. (1984a)

34

65

Ross et al. (1984)

26

17

M. Herlyn et al. (unpublished)

29

23

Donoso et al. (1986)

17

41

Morgan et al. (1984)

Reference

~

HLA-DR

P97

~

13-17, 377 ME31.3, Me9545 ME9-61, MED63 ME491, 8-1H F11

With the exception of antibody F11, all sera were tested in DDIAs.

2 14

MEENHARD HERLYN ET AL.

IV. Shed Tumor-Associated Antigens and Host Response

Antibody responses in cancer patients to autologous tumors have been detected infrequently. When found, they have generally been of insufficient titer to resolve questions of specificity. Old (1981) has demonstrated reactivity in serum of one-third of melanoma patients against cultured autologous tumor cell surface antigens. The antigens described have been divided into three classes: (1)antigens restricted to a tumor of a single individual, (2) antigens shared among tumors of a similar histotype or ontogeny, and (3)antigens shared among nonneoplastic and neoplastic cells. Human MAbs derived by transformation of lymphocytes with Epstein-Barr virus have recently been used to define melanoma-associated gangliosides GD2 and GM2 (Tai et al., 1984; Irie et al., 1982), indicating the immunogenicity of sialogangliosides in melanoma patients. Other human antitumor MAbs derived by fusion of human lymphocytes with human lymphoblastoid cells, human, or mouse myeloma cells detect mainly intracellular antigens (Houghton et al., 1983). These studies could explain previous reports of a very high (90%) occurrence of antinuclear, antinucleolar, and anticytoplasmic antibodies in patients with malignant melanoma (Thomas et al., 1983). Presence of shed TAAs in sera of cancer patients may have been masked by the formation of antigen-antibody complexes. Unfortunately, except for few studies (see below), most investigations were based primarily on the isolation of entire antigen-antibody (immune) complexes (CICs) (see Theophilopoulos and Dixon, 1979, for review) without identification of the antigen and, particularly, comparison to the antigens expressed by cancer cells. More recently, a different approach to studying the humoral immune response to circulating TAAs was taken by Kirkwood and Vlock (1984), who dissociated antigen and antibody complexes as originally described by Sjogren and co-workers (1971). Sera in acid were filtered through a 100,000-MW exclusion membrane that prevented immunoglobulins and other high-molecular-weight serum components from passing through. This procedure has enhanced detection of IgG binding to cultured autologous melanoma cells in 9 out of 10 patients. Antibodies from acid-dissociated serum bound to 8 of 9 melanoma cell lines tested, indicating that the antigens present in CICs were similar to those present on melanoma cells. Binding of antibodies to target cells was blocked by (1)reassociating with the filtered eluate using the antibody present in the retained fraction, (2) spent medium of autologous melanoma cultures, and (3) untreated serum (Vlock and

SHEDDING OF TUMOR-ASSOCIATED ANTIGENS

215

Kirkwood, 1985), indicating that the same TAAs are shed in vivo and in vitro. No further attempts were made, however, to characterize the antigens present in CICs. In longitudinal serum studies, antibody titer was shown to reflect the clinical course of the disease (Vlock and Kirkwood, 1985). Interestingly, Vlock and Kirkwood found no correlation with CIC levels using standard Clq-assays for the detection of CICs, indicating that the antibodies in CICs were not complement fixing. Preliminary results from our laboratory indicate that CICs from colorectal and ovarian carcinoma and melanoma patients contain hightiter and high-affinity antibodies with a remarkable specificity for TAAs as shed by the various tumor cells. Reactivity of antibodies to the target cells was blocked by spent medium from cultures of the same cells, suggesting that antigen(s) present in CICs in patients’ sera are shed by tumor cells in vitro and in vivo.

V. Conclusions

MAbs have detected previously undefined circulating TAAs in sera of patients with various solid human tumors. For established tumor markers, MAbs are replacing the use of polyclonal antisera. It is expected that new tumor markers will become available that may serve, when necessary, as additional diagnostic tools for each tumor type. To date, MAb-defined tumor markers are available for pancreatic and ovarian carcinoma that are elevated in more than 80%of patients’ sera. For other tumors, combinations of assays will need to be developed. All serodiagnostic procedures lack sensitivity for detection of early primary cancer, but they have proved to be very useful in the detection of recurrences and in monitoring established disease. High-molecular-weight, mucin-type structures are the most promising serodiagnostic tumor markers regarding specificity and sensitivity in detection. With only few exceptions all recently developed markers are highly glycosylated structures that may carry both tumor-associated and normal cell-associated determinants. A combination of MAbs with different specificities can be used for their detection to increase sensitivity. The majority of MAbs binding to mucin-type antigens are defining carbohydrate structures. Few serodiagnostic tumor markers are known that are either low-molecular-weight proteins or that circulate as glycolipids. The availability of MAbs will increase studies on the pathophysiology of antigen shedding by tumor cells in vitro and in vivo since little

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is known about this phenomenon. It appears that shed antigens are more immunogenic in cancer patients leading to the formation of immune complexes. Studies for the detection, identification, and characterization of TAAs that are immunogenic in patients should be encouraged in order to improve our understanding of immunopathological phenomena in tumor surveillance. Such studies may also help to detect antigens relevant for the diagnosis of early stages of primary cancer and for assessing the risk of metastasis.

ACKNOWLEDGMENTS These studies were, in part, supported by Grants CA-25874 and CA-10815 from the National Institutes of Health and Grant IM-402 from the American Cancer Society.

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Schwartz, R., Momburg, F., Moldenhauer, G., Dorken, B., and Schirrmacher, V. (1985). Int. J . Cancer 35,245-250. Sears, H. F., Herlyn, M., DelVillano, B., Steplewski, Z., and Koprowski, H. (1982).J . Clin. Immunol. 2, 141-149. Sears, H. F., Herlyn, M., Engstrom, P. F., Shen, J. W., Atkinson, B., and Koprowski, H. (1985).Am. J . Clin. Oncol. 8, 108-117. Shi, Z. R., Tsao, D., and Kim, Y. S. (1983). Cancer Res. 43,4045-4049. Shimano, T., Loor, R. M., Papsidero, L. D., Kuriyama, M., Vincent, R. G., Nemoto, T., Holyoke, E. D., Berjian, R., Douglas, H. O., and Chu, T. M. (1981). Cancer 47, 1602-1613. Shimano, T., Matsuura, N., Tatsuta, M., Kobayashi, T., and Mori, T. (1985).Annu. Meet. Int. SOC.Oncodev. Biol. Med., 13th, Paris, 9/10-13 p. 62. Sjogren, H. O., Hellstrom, I., Bansal, S. C., and Hellstrom, K. E. (1971).Proc. Natl. Acad. Sci. U.S.A. 68, 1372-1375. Staab, H. J., Brunnendorf, T., Hornung, T., Anderer, F. A,, and Kieninger, G. (1985). Klin. Wochenschr. 63, 106-115. Stacker, S . A., Thomson, C., Riglar, C., and McKenzie, I. F. C. (1985). J . Natl. Cancer Inst. 75, 801-811. Steplewski, Z., Chang, T. H., Herlyn, M., and Koprowski, H. (1981). Cancer Res. 41, 2723-2727. Steplewski, Z., Herlyn, M., Blaszczyk, M., and Koprowski, H. (1983).J . Immunol. Methods 62,73-78. Steplewski, Z., Blaszczyk, M., Herlyn, D., Herlyn, M., and Koprowski, H. (1985). In “International Workshop on Monoclonal Antibodies and Breast Cancer” (R. L. Ceriani, ed.), pp. 134-149. Nijhoff, Hingham, Massachusetts. Tagliabue, E., Menard, S., Torre, G. D., Barbanti, P., Mariani-Costantini, R., Porro, G., and Colnaghi, M. I. (1985). Cancer Res. 45, 379-385. Tai, T., Cahan, L. D., Paulson, J. C., Saxton, R. E., and hie, R.F. ( 1 9 8 4 ) ~Natl. . Cancer Inst. 73, 627-633. Theofilopoulos, A. N., and Dixon, F. J. (1979).Adv. Immunol. 28, 89-220. Thomas, P. J., Kaur, J. S., Aitcheson, C. T., Robinson, W. A., and Tan, E. M. (1983). Cancer Res. 43, 1372-1380. Thomson, R. E., Hewitt, C. R., Piper, D. J.. Hansen, W. P., Rubin, R. H., Tolkoff-Rubin, N. E., Barrett, M. C., and Nelles, M. J. (1985). Clin. Chem. 31, 1833-1837. Thurin, J., Herlyn, M., Hindsgaul, O., Karlsson, K.-A., Stromberg, N., Elder, D. E., Steplewski, Z., and Koprowski, H. (1985).J . Biol. Chem. 260, 14556-14563. Vlock, D. R., and Kirkwood, J. M. (1985).J . Clin. Invest. 76, 849-854. Wang, M. C., Valenzuela, L. A., Murphy, G. P., and Chu, T. M. (1979). Inoest. Urol. 17, 159-163. Wang, M. C., Kuriyama, M., Papsidero, L. D., Loor, R. M., Valenzuela, L. A., Murphy, G . P., and Chu, T. M. (1982). Methods Cancer Res. 19, 179-197. Yachi, A., Imai, K., Fujita, H., Moriya, Y., Tanda, M., Endo, T., Tsujisaki, M., and Kawaharada, M. (1984).J . Immunol. 132,2998-3004. Yachi, A., Imai, K., Endo, T., and Takahashi, A. (1985).Annu. Meet. Int. SOC. Oncodeu. Biol. Med., 13th, Paris, 9110-13 p. 67. Yamaguchi, N., and Kawai, K. (1983). Gastroenterology 18, 428-435. Yoshikawa, T., Nishida, K., Tanogawa, M., Fukomoto, K., Kondo, M. (1985). Digestion 31,67-76.

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NEW CLASSES OF TUMOR PROMOTERS: TELEOCIDIN, APLYSIATOXIN, AND PALYTOXIN Hirota Fujiki and Takashi Sugimura National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104, Japan

1. Introduction

Recent studies on phorbol esters at the cellular, biochemical, and molecular levels have provided information on the mechanism of tumor promotion. Because of the importance of human carcinogenesis, it has been necessary to conduct new studies on tumor promotion with tumor promoters other than phorbol esters. When we began studies in 1978, we had the working hypothesis that there could be other potent tumor promoters in the environment that are structurally different from 12-0-tetradecanoylphorbol-13-acetate(TPA) (Hecker, 1967; Van Duuren, 1969) but biologically as active as TPA. Using a short-term screening system for tumor promoters, we soon found two new classes of potent tumor promoters: teleocidin and aplysiatoxin (Fujiki et al., 1981, 1983b, 1984d; Sugimura, 1982; Fujiki and Sugimura, 1983). In previous publications we have called the teleocidin class indole alkaloids and the aplysiatoxin class polyacetates (Fujiki et al., 1981, 1982b, 1983a). However, it now seems more suitable to use the terms teleocidin class and aplysiatoxin class, because several additional new tumor promoters related to teleocidin and aplysiatoxin have also been found. For example, biosynthetic intermediates of the teleocidin class have been isolated from Streptoverticillium (Irie et al., 1984; Sakai et al., 1984), and analogs of teleocidin have been obtained by chemical syntheses (Endo et al., 1984). Tumor promoters of the teleocidin and aplysiatoxin classes are classified as TPA-type tumor promoters because they have the same biological and biochemical effects and have almost the same specific activities as TPA, although they differ in structure from TPA, and because they bind to the same phorbol ester receptors as TPA (Fujiki et al., 1984d). Moreover, TPA, teleocidin, and aplysiatoxin were all found to activate protein kinase C in vitro (Castagna et al., 1982; Fujiki et al., 1984e). 223 ADVANCES IN CANCER RESEARCH, VOL. 49

Copyright 0 1987 by Academic Press, Inc

All ixghts of reproduction In any form reserved

224

HIROTA FUJIKI AND TAKASHI SUGIMURA

Working from biochemical information on the mechanism of tumor promotion induced by TPA-type tumor promoters, we tried to find antitumor agents that inhibit the effect of tumor promoters on mouse skin. A calmodulin antagonist, N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7), markedly inhibited the tumor-promoting activity of teleocidin (Nishino et al., 1984~).Similarly, quercetin, glycyrrhetic acid, and berberine sulfate inhibited the promotion of tumor formation by TPA and teleocidin in vivo (Kato et al., 1983; Nishino et al., 198413, 1984d, 1986a, 1986b; Fujiki et al., 1986a). Studies along these lines might suggest new approaches to the study of human cancer prevention. In studies on new tumor promoters, we also paid attention to compounds that did not respond like TPA-type tumor promoters to our short-term screening system. In this way palytoxin was found to be a non-TPA-type tumor promoter that does not bind to phorbol ester receptors (Fujiki et al., 1984d). Palytoxin is a water-soluble toxin of a coelenterate of the genus Palythoa (Moore and Bartolini, 1981; Uemura et al., 1981). This article reviews studies on these two new classes of TPA-type tumor promoter, teleocidin and aplysiatoxin, and briefly describes findings on the non-TPA-type tumor promoter, palytoxin. II. History and Background of Discoveries of Teleocidin, Aplysiatoxin, and Palytoxin

Our working hypothesis that there could be other potent tumor promoters that are structurally different from TPA but biologically as active as TPA was proved by the discovery of dihydroteleocidin B, which has potent tumor-promoting activity (Fujiki et al., 1979, 1981; Sugimura et al., 1982). Dihydroteleocidin B is a catalytically hydrogenated derivative of teleocidin B (Fig. 1).It had already been reported to be a strong skin irritant, and it is present in mycelia of Streptomyces mediocidicus (Takashima and Sakai, 1960; Takashima e t al., 1962). It was named “teleocidin” because it is toxic to the teleost fish Oryzias latipes. Fortunately, we tested the activity of dihydroteleocidin B, kindly provided by Dr. M. Takashima. Our short-term screening system consists of three tests. The first test is on irritation of mouse ear, the second on induction of ornithine decarboxylase (ODC) in mouse skin, and the third on adhesion of human promyelocytic leukemia cells (HL-60). Compounds are subjected to these three tests successively,

NEW CLASSES OF TUMOR PROMOTERS

Dihydroteleocidin B

225

Teleocidin B

OCO(CH21nCH,

0 20

TPA

Aplysiatoxin

k

Teleocidin A Lyngbyatoxin A

Debromoaplysiatoxin

FIG. 1. Structures of various tumor promoters. The C-14 of teleocidin A shown in a dashed circle has two variable positions, R and S . One isomer of teleocidin A corresponds to lyngbyatoxin A. The numbering system of teleocidin A is not the same as that of teleocidin B.

and dihydroteleocidin B gave positive results in each. Finally, its tumor-promoting activity was investigated in a long-term two-stage mouse skin carcinogenesis experiment. Apart from TPA, dihydroteleocidin B was the first tumor promoter found that gave positive results in all four successive tests (Table I).

TABLE I EFFECTSOF Vmrous TUMOR PROMOTERS

Tumor promoter

Irritant test, ID'$$' (nmol/ear)

Induction of ODC (nmol COz/mg proteid5.0 pg compoundl30 min)

Adhesion of HL-60 cells, EDmb (nglml)

Dihydroteleocidin B Teleocidin Lyngbyatoxin A Aplysiatoxin Debromoaplysiatoxin TPA

0.017 0.008 0.01 1 0.005 0.005 0.016

1.55 1.89 2.05 2.15 2.05 1.45

0.3 4.0 7.0 2.0 180.0 1.5

Inhibition of specific binding of 3H-TPA, EDSb

Tumorbearing mice in week 30

(nM)

(%)

24.0 4.5 48.0 6.6 8.0 4.2

100.0' 87.0d 73.3' 71.u 92.7"

ID:, Irritant dose causing reddening of the ears of 50% of the mice after 24 hr (Hecker, 1971). EDrn, Effective dose giving 50% of the maximal response. 2.5 pg twice a week from weeks 1 to 30. 3.0 pg twice a week from weeks 1 to 30. 2.7 pg twice a week from weeks 1 to 30. f 2.4 pg twice a week from weeks 1 to 30. b

9o.oc

NEW CLASSES OF TUMOR PROMOTERS

227

Since dihydroteleocidin B is more easily crystallized than teleocidin B, its tertiary structure was determined by X-ray crystallography of the monobromoacetate (Harada et al., 1966). The structure of teleocidin B was then deduced from that of dihydroteleocidin B (Fig. 1). Teleocidin B, with a molecular weight of 451, has the unique structure of an indole system and a nine-membered lactam ring. In 1979, Moore’s group reported the structure of lyngbyatoxin A, with a molecular weight of 437, isolated from the Hawiian blue-green alga, Lyngbya majuscula (Cardellina et al., 1979) (Fig. 1). Since lyngbyatoxin A has a structure similar to teleocidin B and, like teleocidin B, has an irritant effect on human skin, we, in collaboration with Dr. R. E. Moore, tested lyngbyatoxin A in our short-term screening system for tumor promoters and finally showed that it has potent tumor-promoting activity in two-stage mouse skin carcinogenesis (Fujiki et al., 1984a) (Table I). During experiments with dihydroteleocidin B and lyngbyatoxin A, we tried to purify teleocidin B, the parent compound of dihydroteleocidin B from a methanolic extract of Streptomyces mediocidicus. The purified material, which gave a single spot on thin-layer chromatography, was shown by high-performance liquid chromatography (HPLC) to be a mixture of teleocidin A, with a molecular weight of 437, and teleocidin B, with a molecular weight of 451. We named this mixture of teleocidin A and teleocidin B “teleocidin.” “Teleocidin” was as strong a tumor promoter as TPA (Fujiki et al., 1981, 1982a) (Table I). Moreover, we found that teleocidin A consists of two isomers and teleocidin B of four (Fujiki and Sugimura, 1983; Fujiki et al., 1984d). Although teleocidin A was found in Streptomyces mediocidicus in 1962 (Takashima et al., 1962), its chemical structure was not determined. In August 1980, there was a severe outbreak of swimmer’s itch on the windward side of Oahu Island. Moore (1982) isolated aplysiatoxin and debromoaplysiatoxin from the marine blue-green alga, Lyngb ya majuscula, as the causative agents of this seaweed dermatitis. Aplysiatoxin and debromoaplysiatoxin, a debrominated form of aplysiatoxin, have an acetogenic, phenolic bislactone structure (Fig. 1). These two toxins were originally found in the digestive gland of the sea hare, Stylocheilus longicauda, and their chemical structures were determined by Kato and Scheuer (1974). Since these toxins were also known to be strongly dermonecrotic, we examined them in our shortterm screening system for tumor promoters and found that they were new tumor promoters. They are classified into a third class of potent tumor promoters distinct from the phorbol ester and teleocidin classes

228

HIROTA FIJJIKI AND TAKASHI SUGIMIJRA

(Fujiki et al., 198213) (Table I). We then carried out structure-function studies on 12 aplysiatoxin derivatives and investigated the tumorpromoting activities of 6 of them in two-stage mouse skin carcinogenesis (Suganuma et al., 1984; Moore, 1984). Seaweed dermatitis was also reported on Okinawa Island in Japan (Hashimoto et al., 1976). Aplysiatoxin and debromoaplysiatoxin were isolated from the marine blue-green alga, Lyngb ya majuscula, collected at Gushigawa Beach on Okinawa Island (Fujiki et al., 1985a). They are supposed to be the causative agents of swimmer’s itch in Japan. The two new classes of potent tumor promoters, teleocidin and aplysiatoxin, were discovered using our short-term screening system. The first important indication of the existence of these new potent tumor promoters was their effect in causing strong irritation of human skin. Since teleocidin and aplysiatoxin exerted the same biological activities as TPA through the same receptor as TPA (Umezawa et al., 1981; Schmidt et al., 1983; Fujiki et al., 198313; Fujiki and Sugimura, 1983; Sugimura and Fujiki, 1983), we classified tumor promoters of the teleocidin and aplysiatoxin classes in the larger category of TPAtype tumor promoters (Fujiki et al., 1984d). When we realized that our short-term screening system could detect only new TPA-type tumor promoters, we turned our attention to nonTPA-type tumor promoters. For this purpose we looked for compounds that did not give positive results in all the short-term screening tests. Palytoxin is a strong skin irritant, but it does not induce ODC activity in mouse skin 4 hr after its application or induce HL-60 cell adhesion. However, it is a tumor promoter in two-stage carcinogenesis in mouse skin. Therefore, palytoxin appears to be a potent non-TPAtype tumor promoter that does not bind to phorbol ester receptors. Recently, we demonstrated that both TPA-type and non-TPA-type tumor promoters have common effects in inducing biological activities such as irritation of mouse ear and production of prostaglandin Ez by rat macrophages (Ohuchi et al., 1985).We began our investigations by screening for new tumor promoters other than TPA and consequently found both TPA-type and non-TPA-type tumor promoters, which will serve as useful tools to obtain important information on tumor promotion in human cancer. 111. Teleocidin Tumor Promoters

A. TELEOCIDIN A AND LYNGBYATOXIN A

When “teleocidin,” isolated from Streptomyces mediocidicus, was subjected to HPLC on LS-410 ODS SIL in 70% acetonitrile, one peak

NEW CLASSES OF TUMOR PROMOTERS

229

of teleocidin A and two peaks of teleocidin B were obtained. Teleocidin A and teleocidin B were identified by their UV absorptions and other properties as reported by Takashima and Sakai in 1960. Teleocidin A was further separated into two fractions by HPLC on an LS-310 SIL column with n-hexane-chloroform-isopropanol(85 : 10 : 5) as solvent. The two peaks of teleocidin A were named A-1 and A-2 in order of their elution. Since they had identical UV absorptions, lH-nuclear magnetic resonance (lH-NMR) and mass spectra, and molecular weights, we concluded that teleocidin A consists of two isomers (Fujiki et al., 1983b; Fujiki and Sugimura, 1983) (Fig. 1). Lyngbyatoxin A was purified from the blue-green alga, Lyngbya mujuscula, collected at Kahala beach on Oahu Island in Hawaii, by a slight modification of the procedure reported by Cardellina et al. (1979). Details of the conditions for isolation of lyngbyatoxin A were reported previously (Fujiki et al., 1984a). Purified lyngbyatoxin A was subjected to HPLC on an LS-310 SIL column, which separates teleocidin A into two fractions, A-1 and A-2. Interestingly, lyngbyatoxin A was identical to teleocidin A-1 in its elution pattern on HPLC and also in its molecular weight, UV absorption, and 'H-NMR and mass spectra (Fujiki et al., 1983b). From these results it was concluded that teleocidin A-1 has the same structure as lyngbyatoxin A with a molecular weight of 437 and that teleocidin A consists of two isomers, 14R and 14S, because C-14 of teleocidin A is a chiral carbon (Fig. 1).The numbering system for teleocidin A was adopted from that for lyngbyatoxin A (Cardellina et aE., 1979). Recently, we, in collaboration with Dr. S. Sakai's group, determined the absolute configuration of teleocidins A-1 (lyngbyatoxin A) and A-2 by chemical degradation, including ozonolysis. The work revealed that teleocidin A- 1 corresponds to C14R-teleocidin A, and teleocidin A-2 to C-14S-teleocidin A, respectively (Sakai et al., 1986a). It is interesting that the same compound, teleocidin A-1, is present in Streptomyces and in a blue-green alga. The effects and tumor-promoting activities of the two teleocidin A isomers are summarized in Table 11.

B. TELEOCIDIN B AND DIHYDROTELEOCIDIN B Teleocidin B, with a molecular weight of 451, was separated into four fractions by HPLC on LS-410A ODS SIL in 75% methanol. The four peaks of teleocidin B were named B-1, B-2, B-3, and B-4, in order of their elutions, because they were thought to be isomers based on their UV absorptions and IH-NMR and mass spectra. When the chemical structure of teleocidin B was deduced from X-ray studies of dihy-

TABLE I1 EFFECTSOF Two TELEXIDIN A ISOMERS AND THREE TELEXIDINB ISOMERS

Teleocidin isomer A- 1 A-2 B-2 B-3 B-4

Irritant test, ID: (nmol/ear)

Induction of ODC (nmol COz/mg proteinlll nmol compoundl30 min)

Adhesion of HL-60 cells, ED% (nglml)

Inhibition of specific binding of "-TPA, K , [(M) x 10-91

Tumorbearing mice in week 30b

0.032 0.016 0.032 0.016 0.016

3.31 3.80 4.70 4.21 3.97

1.24 1.45 1.59 1.61 1.25

3.2 2.1 2.9 3.4 2.7

86.6 86.7 80.0 86.7 91.7

Minimum dose of compound causing reddening of mouse ear after 24 hr. 2.5 pg of compound per application twice a week.

(90)

231

NEW CLASSES OF TUMOR PROMOTERS

droteleocidin B monobromoacetate (Harada et al., 1966),the presence of four teleocidin B isomers was not reported. More recently, the four teleocidin B isomers were each obtained in a crystalline state. The structures of teleocidins B-1 and B-4 were determined from X-ray analyses of des-0-methylolivoretin B and olivoretin D, respectively (Hitotsuyanagi et ul., 1984a). Olivoretins B and D were isolated from Streptouerticillium oliuoreticuli, and their chemical structures were elucidated (Hitotsuyanagi et al., 1984b; Sakai et ul., 1984) (Fig. 2). In collaboration with Dr. S. Sakai's group it was found that teleocidin B-1 is identical to des-0-methylolivoretin By and teleocidin B-4 to olivoretin D (Fig. 2). Olivoretin D is identical in structure to teleocidin B, which was used for studies on dihydroteleocidin B monobromoacetate in 1966. Therefore, teleocidin Bywhich Takashima and Sakai isolated from Streptornyces in 1960, was shown to be teleocidin B-4. Thus, it is thought that the crystalline form of dihydroteleocidin B monobromoacetate was fortuitously obtained as a major crystalline form of the four teleocidin B isomers. When Sakai and associates (1984) elucidated the structure of olivoretin D by X-ray analysis, they noticed that the first structure formula of dihydroteleocidin B monobromoacetate, published in

Olivoretin A

Olivoretin B

Olivoretin D Des-O-methylolivoretin A Teleocidin 8-4

Des-O-methylolivoretin B Teleocidin B-1

Olivoretin C

I

Des-0-methylolivoretin C

FIG. 2. Structures of olivoretins A, B, C, and D. Olivoretin D is identical to one teleocidin B isomer, B-4.

232

HIROTA FUJIKI AND TAKASHI SUGIMURA

1966, was erroneous with regard to the relative stereochemistries of the four chiral centers. Figures 1 to 3 in this article depict the correct structural formulas of teleocidin B and dihydroteleocidin B. The structures of teleocidins B-2 and B-3 were elucidated by studies on 13C-NMRspectra (Hitotsuyanagi et al., 1984a).From the results it was concluded that the four teleocidin B isomers are C-19,C-22diastereomers; (19S,22R)-teleocidin B for B-1, (19R,22S)-teleocidin B for B-2, (19S,22S)-teleocidin B for B-3, and ( 19RY22R)-teleocidinB for B-4 (Fig. 3). Although C-l9,C-22-diastereomers were formerly called C-14,C-17-diastereomers (Fujiki et al., 1984d1, we recently changed the numbering of carbon atoms according to the revised numbering system for teleocidin Bybecause both Sakai’s group and Koshimizu’s group independently introduced the proper numbering system for teleocidin B (Hitotsuyanagi et al., 1984a; Irie et al., 1984). The effects of tumor-promoting activities of teleocidins B-2, B-3, and B-4 are summarized in Table 11. C. OLIVORETINS A, ByC, AND D From mycelia of Streptoverticillium olivoreticuli, Sakai and associates isolated olivoretins A, Byand C with a molecular weight of 465

C - l g S , C-22R Teleocidin B B- 1

C-I9R, C - 2 2 s Teleocidin B 6-2

C - l g S , C - 2 2 s Teleocidin B

C-l9R, C-22R Teleocidin B

8-4 FIG.3. Structures of teleocidins B-1, B-2, B-3, and B-4.

8-3

233

NEW CLASSES OF TUMOR PROMOTERS

and olivoretin D with a molecular weight of 451. The first three olivoretins are all 0-methylated at a primary hydroxyl group and were inactive in the short-term screening system for tumor promoters. These three olivoretins could not bind to phorbol ester receptors in cell membranes and were not assumed to be tumor promoters (Horiuchi et d.,1984) (Table 111). As Fig. 2 shows, the isopropyl group at C-19and the vinyl group at C-22in olivoretin B are located in positions opposite from those in olivoretin C.These structural differences between olivoretin B and olivoretin C resulted in marked differences in several biological activities of des-0-methylolivoretin B (teleocidin B-1) and des-o-methylolivoretin C. Des-0-methylolivoretin C showed slightly weaker activity than des-0-methylolivoretin B in the four tests; irritation of mouse ear, induction of ODC, induction of adhesion of HL-60 cells, and inhibition of specific binding of 3H-labeled TPA (Table 111). Similarly, des-0-methylolivoretin C had slightly weaker tumor-promoting activity than des-0-methylolivoretin B (teleocidin B-1) in two-stage mouse skin carcinogenesis (Ninomiya et al., 1986). Des-0-methylolivoretin C was recently isolated from Streptomyces mediocidicus as a naturally occurring compound (Sakai et al., 1986b). Biologically active olivoretin D was shown to be des-0-methylolivoretin A from its 13CNMR and lH-NMR spectra (Sakai et al., 1984). As described previously, olivoretin D (des-0-methylolivoretin A) is identical to teleocidin B-4 (Fig. 2).Since the three 0-methylated olivoretins, A, B and C, are all inactive, the free primary hydroxyl group of des-0-methylolivoretins (teleocidin B isomers) is necessary for biological activity. TABLE I11 EFFECTS OF OLIVORETINS ~~

Olivoretin Olivoretin A Olivoretin B Olivoretin C Olivoretin D (teleocidin B-4) Des-0-metbylolivoretin B (teleocidin B-1) Des-0-methylolivoretin C

Irritant test (100 ng) -

-

+++ +++

+

Induction of ODC (nmol C02/mg protein/5.0 pg compound/30 min)

Inhibition of Adhesion of specific binding HL-60 cells, of 3H-TPA, ED50 ED50 (nM) (nM)

0.03 0 0 3.97

>2 x 104 >2 x 104 >4 x 103 7.8

>1 x 104 > i x 104 >1 x 104 7.5

4.13

8.4

30.0

1.45

26.6

120.0

234

HIROTA FUJIKI AND TAKASHI SUGIMURA

Recently, Koshimizu and associates also isolated olivoretins A, B, C, and D from Streptoverticillium blastomyceticum (Irie et al., 1985).

D. BIOSYNTHETIC INTERMEDIATES OF TELEOCIDINS AND OLIVORETINS, ( -)-INDOLACTAM-V Teleocidins and biologically active des-0-methylolivoretins have the same modified dipeptide structure, which consists of an indole system and a nine-membered lactam ring. This structure was named “( -)-indolactam-V,” based on the constituents of the two molecules, L-valine with the lactam ring structure and L-tryptophan (providing the suffix V) (Fig. 4) (Fujiki et al., 1984b). (-)-Indolactam-V possesses the S,S configuration at the isopropyl residue and the hydroxymethyl group due to the peptide structure of the two L-amino acids. Shudo and associates synthesized four indolactam-Vs, (&)-indolactam-Vand (+)-epiindolactam-V, from (f)-4-nitrotryptophanol (Endo et al., 1982, 1984) (Fig. 4). Of the four, only (-)-indolactam-V bound to phorbol ester receptors in cell membranes and induced both ODC activity in mouse skin and adhesion of HL-60 cells, although its effects were much weaker than those of teleocidins A and B. These results indicated that the S,S configuration of (-)-indolactam-V in teleocidins A and B and des-0-methylolivoretins is necessary for activity (Fujiki et al., 1984b; Horiuchi et al., 1984).

-. .

( -1-lndolactam-V

(+)-lndolactam-V

H N-methyl-L-valylL-tryptop hanol

-w

Y CH3-N+N\I’1

H ‘OH

H

(-1-epi-lndolactam-V

H

(+)-epi-lndolactam-V

FIG.4. Structures of four indolactam-Vs and the possible precursor of (-)-indolactam-V, N-methyl-L-valyl-L-tryptophanol.

NEW CLASSES OF TUMOR PROMOTERS

235

Recently, a large quantity of (-)-indolactam-V was obtained from the culture broth of Streptoverticillium blastomyceticum as a possible biosynthetic intermediate of teleocidin B (Irie et al., 1984). In collaboration with Dr. K. Koshimizu’s group, we have reported that (-)-indolactam-V has weak tumor-promoting activity in two-stage carcinogenesis in mouse skin. Its potency is compatible with its low potency in the short-term screening system (Fujiki et al., 1985b) (Table IV). Therefore, a large hydrophobic domain attached to the (-)indolactam-V molecule is important for induction of biological activities and for expression of tumor-promoting activity. Sakai and associates identified a possible precursor of (-)-indolactam-V, N methyl-L-valyl-L-tryptophanol, in Streptoverticillium ( S . Sakai, personal communication) (Fig. 4).Since N-methyl-L-valyl-L-tryptophan01 is biologically inactive and does not bind to phorbol ester receptors (H. Fujiki et al., unpublished results), (-)-indolactam-V probably becomes biologically active after a nine-membered lactam ring is formed from its possible precursor, N-methyl-L-valyl-L-tryptophanol.

E. TWO-STAGE CARCINOGENESIS WITH TELEOCIDINS Carcinogenesis was initiated by a single application of 100 p g of 7,12-dimethylbenz[a]anthracene(DMBA) to the skin of the back of 8week-old female CD-1 mice (Fujiki et al., 1981). After 1 week, one teleocidin compound, in the amount shown in the footnotes to Tables I and 11, was applied twice a week until week 30. Three control groups were treated with the same amount of DMBA, a teleocidin, and the solvent acetone, alone, respectively. Each group consisted of 15 mice. The following parameters were used for evaluation of tumorpromoting activity: the percentage of tumor-bearing mice, the average number of tumors per mouse, and histopathological findings on tumors in week 30 of tumor promotion (Fujiki et al., 1982a).As Tables I and I1 show, the percentages of tumor-bearing mice in the groups treated with DMBA plus dihydroteleocidin B, teleocidin (93% teleocidin A and 7% teleocidin B), lyngbyatoxin A, teleocidins A-1, A-2, B-2, B-3, and B-4 were similar to that in the group treated with DMBA plus TPA (Fujiki et al., 1984d). Results for TPA are averages of those in three experiments with 2.5 pg of TPA. Teleocidin B-1 was not tested because of its limited availability due to low content in Streptomyces mediocidicus. However, we were able to show that des-0methylolivoretin B (teleocidin B-1) has tumor-promoting activity as strong as the other teleocidin A and B isomers (Ninomiya et al., 1986).

TABLE IV

EFFECTSOF IND IN DO LAC TAM-^

AND

TELEOCIDIN A

(nmol C02/mg, proteid5.0 pg compound/30 min)

Adhesion of HL-60 cells, ED50 (ng/ml)

Inhibition of specific binding of 3H-TPA, ED50 (nM)

Tumorbearing mice in week 30 (%)

0.43 3.65

28.0 1.4

1,100 7.5

29" 87

Induction

of ODC Tumor promoter

Irritant test (1 p g )

(-)-Indolactam-V Teleocidin A

++++

-

12.5 pg of (-)-indoiactam-V. 2.5 pg of teleocidin A per application.

NEW CLASSES OF TUMOR PROMOTERS

237

(-)-Indolactam-V had lower tumor-promoting activity than the other teleocidins (Fujiki et al., 1985b) (Table IV). It is noteworthy that the average number of tumors per mouse was 3-5 in the group treated with DMBA plus a teleocidin but 12 in the group treated with DMBA plus TPA. The percentages of squamous cell carcinoma and of papilloma including hyperplasia were 20.5 :79.4 with dihydroteleocidin B, 13.2 : 86.8 with teleocidin, 3.6 :96.4 with lyngbyatoxin A, and 4.0 : 95.5 with TPA (Sugimura et al., 1982; Fujiki et al., 198171982a,1984a,c).Thus teleocidins were as effective as TPA in production of malignancy in skin tumors. The tumor-promoting activities of three doses (1.25,2.5, and 5.0 pg) of dihydroteleocidin B were examined in DMBA-initiated mouse skin. A dose of 2.5 pg caused the greatest increase in the percentage of tumor-bearing mice and resulted in the highest average number of tumors per mouse. The optimal dose of dihydroteleocidin B for tumor promotion coincided with that for ODC induction (Suganuma et al., 1982).

F. ANTITUMORPROMOTERS AGAINST TELEOCIDIN The tumor-promoting activities of croton oil and TPA in mouse skin are inhibited by various compounds (Wattenberg, 1985), such as protease inhibitors (Troll et d., 1970; Hozumi et d.,1972), a vitamin A derivative (Verma et al., 1979), an inhibitor of polyamine synthesis (Takigawa et al., 1982), and an inhibitor of histidine decarboxylase (Umezawa et al., 1983). 13-cis-Retinoic acid (Fujiki et al., 1981),indomethacin as a selective cyclooxygenase inhibitor, and p-bromophenacyl bromide as a phospholipase A2 inhibitor all inhibited ODC induction by teleocidin (Nakadate et al., 1985). Furthermore, treatment with lipoxygenase inhibitors such as BW755C, nordihydroguaiaretic acid, quercetin, and 2,3,5-trimethy1-6-(12-hydroxy-5,10-dodecadiynyl)-174-benzoquinone (AA861) also suppressed ODC induction by teleocidin (Nakadate et al., 1985). Various compounds are reported to inhibit the effects of teleocidin, and we systematically screened for antitumor promoters to develop chemopreventive agents. For this purpose, we tested whether compounds inhibited the acceleration of 32Piincorporation into phospholipid fractions of mouse fibroblasts induced by teleocidin (Nishino et al., 1983). A calmodulin antagonist, N-(6-aminohexyl)-5-chloro-lnaphthalenesulfonamide (W-7), quercetin, glycyrrhetic acid, and berberine sulfate were found by this test and they markedly inhibited the promoting activity of teleocidin on tumor formation in mice initi-

238

HIROTA FUJIKI AND TAKASHI SUGIMURA

ated with DMBA (Nishino et al., 1984b,c,d, 1986a,b; Fujiki et aZ., 1986a). On the other hand, W-7 and trifluoperazine were shown to inhibit phosphorylation of histone by protein kinase C (Wise et al., 1982), which is suggested to serve as a receptor for tumor promoters (Nishizuka, 1984). The mechanism of the antitumor promoting activity of W-7 was examined more precisely by analyzing the effect of W-7 on the phorbol ester receptors in mouse skin. We found that W-7 reduced the number of receptors in mouse skin in a dose- and time-dependent manner (Nishino et al., 1984a).Thus, W-7 probably inhibits the formation of a quaternary complex of the tumor promoter, protein kinase C, Ca2+,and phospholipid (Kikkawa et al., 1983), owing to its interaction with phospholipid. Measurement of the reduction of the number of phorbol ester receptors in mouse skin appears to be a good screening test for anti-tumor promoters (Fujiki et aZ., 1986a; Horiuchi et al., 1986). IV. Aplysiatoxin Tumor Promoters

A. APLYSIATOXIN AND ITS DERIVATIVES A new, third class of tumor promoters, “aplysiatoxin,” showed interesting results on induction of two biological activities of cells (Fujiki et al., 1982b); debromoaplysiatoxin was about 100 times less potent in inductions of HL-60 cell adhesion (ED50 180 ng/ml) and aggregation of human lymphoblastoid cells (NL-3 cells) (ED50 180 ng/ml) than aplysiatoxin (ED50 2.0 and 2.1 ng/ml, respectively). The potency of aplysiatoxin for these two biological activities was similar to that of teleocidin and TPA. However, debromoaplysiatoxin and aplysiatoxin showed the same activity as teleocidin and TPA with regard to irritation of mouse ear and induction of ODC in mouse skin (Table I). Structurally debromoaplysiatoxin differs from aplysiatoxin only in not having a bromine atom in the phenol group; otherwise they both have the structure of an acetogenic, phenolic bislactone (Figs. 1 and 5 ) . A bromine atom of aplysiatoxin is supposed to play a crucial role in inductions of HL-60 cell adhesion and aggregation of NL-3 cells. Debromoaplysiatoxin and aplysiatoxin were first found to be useful compounds for analysis of the significance of biological activities; for example, irritation and ODC induction might be induced by a mechanism different from that of adhesion of HL-60 cells and aggregation of NL-3 cells. Therefore, debromoaplysiatoxin and aplysiatoxin were expected to provide information on whether irritation and ODC

239

NEW CLASSES OF TUMOR PROMOTERS

OH

OH

OH

OH

Oscillatoxin A

Debromoaplysiatoxin

H

H

H

Aplysiatoxin

Br

H

H

Bromoaplysiatoxin

Br

Br

H

Dibromoaplysiatoxin

Br

Br

Br

RI

R2

Anhydrodebromoaplysiatoxin

H

CH,

An hydroaplysiatoxin

Br

CH3

Anhydrooscillatoxin A

H

H

OH

OH

Oscillatoxin B

R,

R2

20-0-methyl-debromoaplysiatoxin

CHI

H

Debromoaplysiatoxin20-acetate

CH3C0

H

Debromoaplysiatoxin20, 30-diacetate

CH3C0 CH3C0

FIG.5. Structures of 12 aplysiatoxin derivatives.

induction are much more closely correlated with tumor-promoting activity than adhesion of HL-60 cells and aggregation of NL-3 cells. From this standpoint, we studied the effects of twelve aplysiatoxin derivatives with bromine atoms in different positions. The structures of these twelve aplysiatoxin derivatives and the numbering system are shown in Fig. 5. In addition to debromoaplysiatoxin (without a bromine) and aply-

240

HIROTA FUJIKI AND TAKASHI SUGIMURA

siatoxin (with one bromine), anhydrodebromoaplysiatoxin and anhydroaplysiatoxin were isolated from Lyngbyu mujuscula collected at Kailua beach, Oahu Island (Kato and Scheuer, 1975). These two anhydro derivatives were formed from the toxins either on chromatography during their purifications or during electron-impact mass spectrometry by facile, acid-catalyzed dehydration between C-3 and C-4 of the acetogenic, phenolic bislactone (Kato and Scheuer, 1976). Whether these anhydro derivatives are naturally occurring compounds is not reported. Mynderse and Moore (1978) isolated oscillatoxin A (31-nordebromoaplysiatoxin, a demethylated form of debromoaplysiatoxin), oscillatoxin B, and anhydrooscillatoxin A from a mixture of Oscillatoria nigroviridis and Schizothrix calcicola, collected at Eniwetok Atoll, Marshall Islands. Three minor bromine-containing compounds isolated from this algal mixture were identified chemically as 21-bromooscillatoxin A, 19,21-dibromooscillatoxin A, and 19-bromoaplysiatoxin (aplysiatoxin with two bromines) (Mynderse and Moore, 1978). It is now possible to obtain bromoaplysiatoxin and dibromoaplysiatoxin (with three bromines) by bromination of debromoaplysiatoxin in aqueous methanol buffered at pH 6.0 with sodium phosphate (Moore et al., 1984). 20-0-Methyldebromoaplysiatoxin was prepared by methylation of debromoaplysiatoxin with diazomethane, and debromoaplysiatoxin-20-acetate and debromoaplysiatoxin-20,30diacetate were formed by acetylation of debromoaplysiatoxin with acetic anhydride and pyridine (Suganuma et al., 1984). The effects of 12 aplysiatoxin derivatives on irritation of mouse ear, induction of ODC activity in the skin of the back of mice, and inhibition of specific binding of 3H-labeled TPA are summarized in Table V. The potencies of irritation and induction of ODC activity by various aplysiatoxin derivatives correlated closely with their affinities to phorbol ester receptors in cell membranes. Furthermore, from the results in Table V the roles of the three hydroxyl groups on C-3, C-20, and C30 in the structure-function relationship could be determined. The C3 hydroxyl group appears to be necessary for biological activity. Derivatization at C-20 and C-30 resulted in reduction in activity, suggesting that C-20 and C-30 hydroxyl groups may also be necessary for maximum activity. Moore and colleagues (1984) elucidated the absolute stereochemistries of aplysiatoxin derivatives including oscillatoxin A by 'H-NMR spectral studies and X-ray crystallographic analyses, Based on these results, inspection of Dreiding models showed that the oxygens on C-27, C-3, and C-30 of aplysiatoxin align with the oxygens on C-3, C-4, and C-20 of TPA, respectively (Moore, 1984) (Fig. 1).It is interesting that the hydroxyl groups on C-30 of aplysiatoxin, C-24 of teleocidin A, C-14 of teleocidin B, and C-20 of TPA

NEW CLASSES OF TUMOR PROMOTERS

24 1

TABLE V EFFECTS OF 12 APLYSIATOXIN DERIVATIVES

Apl ysiatoxin derivative Debromoaply siatoxin Aplysiatoxin Bromoaplysiatoxin Dibromoaplysiatoxin Anh ydrodebromoaplysiatoxin Anhydroaplysiatoxin Anhydrooscillatoxin A Oscillatoxin A Oscillatoxin B 20-O- Methy Idebromoaplysiatoxin Debromoaplysiatoxin-20-acetate Debromoaplysiatoxin-20,30-diacetate TPA a

Irritant test (0.1 nmol)=

+++ ++++ ++ -

++ + +++ ++ +++

Induction of ODC (nmol C02/mg proteint3 nmol compound/30 min)

Inhibition of specific binding of 3H-TPA, K , [(M) x 1041

5.52 5.06 2.27 0.02 0.15 0.30 0.15 1.50 0.05 3.00 4.76 1.49 3.02

2.7 2.6 2.6 1300 210 170 190 3.9 >3200 32.0 15.0 93.0 1.4

Redness was estimated after 24 hr.

play structurally and functionally similar roles in the activity of TPAtype tumor promoters (Fig. 1). Debromoaplysiatoxin differs from aplysiatoxin in having no bromine atom. The presence of a bromine atom results in 100-fold increase in the induction of HL-60 cell adhesion (Table I). Studies were also made on the inductions of HL-60 cell adhesion by four aplysiatoxin derivatives containing various numbers of bromine atoms: debromoaplysiatoxin (without a bromine), aplysiatoxin (with one bromine), bromoaplysiatoxin (with two bromines), and dibromoaplysiatoxin (with three bromines). As Fig. 6 shows, the concentrations of these four aplysiatoxin derivatives necessary for 50% cell adhesion were very different. The order of potency of the compounds was bromoaplysiatoxin > aplysiatoxin > dibromoaplysiatoxin > debromoaplysiatoxin. It will be interesting to determine whether the potencies in inducing HL-60 cell adhesion reflect the tumor-promoting activities of these four aplysiatoxin derivatives.

B. TWO-STAGE CARCINOGENESIS WITH APLYSIATOXIN DERIVATIVES We have reported that debromoaplysiatoxin is considerably weaker than aplysiatoxin as a tumor promoter in mouse skin, judging from the average number of tumors per mouse in week 16 of tumor promotion

242

HIROTA FUJIKI AND TAKASHI SUGIMURA

"

0

1

10

100

1000

Concentration ( nM )

FIG.6. Dose-responses of HL-60 cell adhesion to four aplysiatoxin derivatives containing various numbers of bromine atoms (bromoaplysiatoxin,A; aplysiatoxin, 0 ; dibromoaplysiatoxin, A; debromoaplysiatoxin, 0).

(Fujiki et al., 198213). In this experiment, from 1week after initiation with 100 p g of DMBA, debromoaplysiatoxin or aplysiatoxin was given twice a week at a dose of 0.5 pg (0.85 nmol of debromoaplysiatoxin and 0.74 nmol of aplysiatoxin) for 9 weeks, and then at 1.25 pg (2.11 nmol and 1.86nmol, respectively) until week 30. Figure 7a shows that the percentages of tumor-bearing mice in the groups treated with DMBA plus aplysiatoxin and with DMBA plus debromoaplysiatoxin were 93.0 and 53.0, respectively, in week 30. These differences in vivo are consistent with the observed differences in inductions of HL60 cell adhesion and aggregation of NL-3 cells by debromoaplysiatoxin and aplysiatoxin. In the second experiment, we gave 2.37 pg (4.0 nmol) of debromoaplysiatoxin and 2.69 pg (4.0 nmol) of aplysiatoxin per application throughout the two-stage carcinogenesis experiment. In this case debromoaplysiatoxin and aplysiatoxin had almost the same tumor-promoting activities, as Fig. 7b shows. The total amount of debromoaplysiatoxin needed for tumors in 50% of the mice was 79.8 nmol in the first experiment and 88.0 nmol in the second, whereas the amount of aplysiatoxin for 50% tumor induction was only 44.2 nmol in the first experiment and 92.0 nmol in the second. Therefore, aplysiatoxin acted as a slightly stronger tumor promoter than debromoaplysiatoxin in the first experiment. The requirement for 92.0 nmol of aplysiatoxin in the second experiment might indicate that the doses of 4 nmol

243

NEW CLASSES OF TUMOR PROMOTERS

b

a

- 100

8

DAT0.5ugI0.851 AT 0.5ug(O.74)

+

1.25pgl2.1 11 1.25pgl1.861

..........................

Inmoll

DAT 2.37ug14.01 AT 2.69ua14.01

lnmoll

I

al

.-0

E

n,

.-CL

m

f

50 1

0

E

3

I-

0

0

10

20

30 0

10

20

30

Weeks of promotion FIG. 7. Tumor-promoting activities of debromoaplysiatoxin (DAT, 0)and aplysiatoxin (AT, 0 )in two separate mouse skin carcinogenesis experiments.

applied each time exceeded the optimum dose necessary for expression of biological activity on mouse skin. The results show that the difference in the tumor-promoting activities of debromoaplysiatoxin and aplysiatoxin in the previous experiment is caused by differences in the amounts of the compounds applied and that the difference in the potency of debromoaplysiatoxin and aplysiatoxin in inductions of HL-60 cell adhesion and aggregation of NL-3 cells did not correlate with their tumor-promoting activity. In addition to debromoaplysiatoxin and aplysiatoxin, bromoaplysiatoxin, dibromoaplysiatoxin, oscillatoxin A, and anhydrooscillatoxin A were tested at doses of 4 nmol in the two-stage carcinogenesis experiments on DMBA-initiated mouse skin. The percentages of tumor-bearing mice in the groups treated with DMBA plus debromoaplysiatoxin, aplysiatoxin, bromoaplysiatoxin, dibromoaplysiatoxin, oscillatoxin A, and anhydrooscillatoxin A were 71.4, 73.3, 57.1, 66.7, 53.3,and 0 in week 30, respectively. The tumor-promoting activities of these aplysiatoxin derivatives, except dibromoaplysiatoxin, correlated well with responses in the three biological tests described in Table V. However, their potencies in inducing HL-60 cell adhesion did not reflect their tumor-promoting activities. Dibromoaplysiatoxin, which was synthesized chemically by bromination and does not occur naturally, showed strong tumor-promoting activity in a two-stage car-

244

HIROTA FUJIKI AND TAKASHI SUGIMURA

cinogenesis experiment, although very weak responses in short-term tests. The tumor-promoting activity of dibromoaplysiatoxin suggests that in the initiated mouse skin during long-term experiments it might be metabolized to active aplysiatoxin derivatives such as bromoaplysiatoxin, aplysiatoxin, and debromoaplysiatoxin by splitting off its bromine atom. This possibility must be examined by studying the metabolism of radioactive dibromoaplysiatoxin in mouse skin. Moore recently succeeded in synthesizing [3H]debromoaplysiatoxin by catalytic hydrogenation of aplysiatoxin. Using [3H]debromoaplysiatoxin by catalytic hydrogenation of aplysiatoxin, we showed that the doseresponse curves for the specific binding of [3H]debr~m~aply~iatoxin to a mouse particulate fraction are essentially identical to that for 3Hlabeled TPA and t h a the specific binding of [3H]debromoaplysiatoxin was inhibited by unlabeled TPA, teleocidin, and aplysiatoxin (Moore et al., 1986). It is unknown whether anhydrooscillatoxin A is inactive when administered at over 4 nmol per application. As Table V shows, three anhydro derivatives were 100 times weaker than TPA in inhibiting specific binding of 3H-labeled TPA and were inactive in the irritancy and ODC induction tests. Since the dose-response curves of ODC induction by anhydrooscillatoxin A and anhydrodebromoaplysiatoxin increased steadily with doses of up to 320 nmol, the tumor-promoting activity of anhydrodebromoaplysiatoxin was examined by applications of either 20 or 10 nmol. The percentage of tumor-bearing mice in the groups treated with DMBA plus anhydrodebromoaplysiatoxin were 40.0 and 0, respectively (H. Fujiki, unpublished results). From this result we concluded that anhydrodebromoaplysiatoxin, and possibly other anhydro derivatives, is a very weak tumor promoter but is not inactive like phorbol. V. In Vitro and in Vivo Effects of Teleocidins and Aplysiatoxin Derivatives

When we first studied the effects of dihydroteleocidin B, teleocidin, lyngbyatoxin A, debromoaplysiatoxin, and aplysiatoxin in irritation of mouse ear, induction of ODC, induction of adhesion of HL-60 cells, and also in tumor promotion, we were surprised to find that teleocidins, aplysiatoxin, and TPA all induce the same effects in the in vitro and in vivo assay systems and showed the same specific activities. Since tumor promoters of the teleocidin and aplysiatoxin classes are structurally unrelated to TPA, many investigators in various research fields have studied the effects of these tumor promoters and have

245

NEW CLASSES OF TUMOR PROMOTERS

shown that teleocidins and some aplysiatoxin derivatives have high specific activities like that of TPA in various assay systems (Fujiki and Sugimura, 1983; Fujiki et al., 1983b; Sugimura and Fujiki, 1983). In collaboration with Dr. I. B. Weinstein’s group, we first found that teleocidin inhibits the specific binding of [3H]phorbol-12,13-dibutyrate (3H-PDBu) to cell surface receptors of rat embryo fibroblast cells (FRE-8D) (Umezawa et al., 1981). Schmidt in Hecker’s group showed that teleocidins, debromoaplysiatoxin, and TPA have the same strong capacity to inhibit specific binding of [3H]phorboI-12,13dipropionate (3H-PDPr) to a particulate fraction of mouse skin (Schmidt et al., 1983). We also found that dihydroteleocidin B, teleocidin, lyngbyatoxin A, debromoaplysiatoxin, and aplysiatoxin inhibit the specific binding of 3H-labeled TPA to a particulate fraction of mouse skin in a similar way to cold TPA (Fujiki and Sugimura, 1983; Sugimura and Fujiki, 1983; Fujiki et al., 1984~). For direct measurement of specific binding of 3H-labeled TPA, cold acetone was used as washing solution (Ashendel and Boutwell, 1981; Hergenhahn and Hecker, 1981). These results showed that three structurally different classes of tumor promoters, teleocidin, aplysiatoxin, and phorbol ester, induce the same effects through the same phorbol ester receptors. It is noteworthy that TPA-type tumor promoters such as TPA, teleocidin, and aplysiatoxin all showed the same specific activities in inducing various effects, except for a few that will be described later. In the following sections over 60 biological and biochemical properties induced by teleocidins and some aplysiatoxin derivatives are briefly reviewed and compared with those induced by TPA. A. EFFECTSON CELLS TRANSFORMED BY EBV, ADENOVIRUS, TUMOR VIRUSES

AND

RNA

Teleocidin, like TPA, induced aggregation of NL-3 cells transformed by Epstein-Barr virus (EBV) (Hoshino et al., 1980), colony formation of human umbilical cord blood lymphocytes in soft agar induced by the B95-8 strain of EBV (Hoshino et al., 1981), expression of early antigen (EA) and/or capsid antigen (VCA) of EBV in the EBV genome-carrying cell line C-6 and P3HR-1 cells in the presence of nbutyrate (Yamamoto et al., 1981; Eliasson et al., 1983; Ito et al., 1984), and an increase in EBV DNA replication in parallel with the appearance of viral antigen synthesis (Lin and Smith, 1984). Teleocidin enhanced transformation of a clone of rat embryo fibroblast (CREF) cells by a temperature-sensitive mutant of adenovirus type 5 (H5ts125) and enhanced the cloning efficiency in agar of a clone of H5ts125-trans-

246

HIROTA FUJIKI AND TAKASHI SUGIMURA

formed rat embryo cells designated as E l l , which has a low plating efficiency in agar (Fisher et aZ., 1982). Dihydroteleocidin B enhanced the production of RNA tumor virus, especially Moloney murine leukemia virus (M-MuLV), by a mouse fibroblast cell line, C3H2K, persistently infected with M-MuLV, as well as growth of the C3H2K cells (Hoshino et al., 1983). Teleocidin produced a pronounced but transient enhancement of the synthesis of extracellular viral particles in human cells infected with simian retroviruses of types C or D or with a human cell line-derived type D isolate (PMF virus) (Wunderlich et al., 1985).

B. EFFECTSON DIFFERENTIATION Teleocidins and aplysiatoxin derivatives induced differentiation of HL-60 cells, characterized by increased phagocytosis, increased release of lysozyme, and morphological changes to cells resembling macrophages (Nakayasu et al., 1981).Teleocidin, with TPA, enhanced phospholipid methylation, measured as the activity of phosphatidylethanolamine methyltransferase, in cell lysates of HL-60 but not in a cell variant, which is resistant to differentiation induction by teleocidin or TPA (Hoffman and Huberman, 1982). TPA-resistant cells were also resistant to teleocidin (Huberman et al., 1982). Dihydroteleocidin B completely inhibited adipocyte differentiation induced by treating growth-arrested 3T3 L1 cells with dexamethasone and l-methyl-3isobutylxanthine, whereas TPA inhibited it only about 10% (Shimizu et al., 1983). Of four indolactam-Vs, only (-)-indolactam-V caused up to 70% inhibition of adipose conversion of ST-13 murine preadipocytes (Sato et al., 1985). C. EFFECTSON DNA Recently, important evidence has accumulated that tumor promoters have not only epigenetic but also genetic effects in cells (Klein and Klein, 1984).Varshavsky (1981a) found that TPA enhances amplification of the dihydrofolate reductase (DHFR) gene in methotrexateresistant mouse cell lines. Shimke’s group showed that TPA enhances the UV-induced increase in DHFR gene amplification (Tlsty et aZ., 1982). Varshavsky (1981b) proposed that illegitimate reinitiation of replication is the primary event in tumor promotion. Three structurally different potent tumor promoters, dihydroteleocidin By aplysiatoxin, and TPA, enhanced the frequency of appearance of cadmium-resistant Chinese hamster lung cells when the cells were

NEW CLASSES OF TUMOR PROMOTERS

24 7

exposed to cytotoxic levels of CdC12. The resistant cells overproduced metallothionein I (MTI) mRNA and had an amplified MTI gene (Hayashi et al., 1983). Teleocidin induced the appearance of smaller extrachromosomal circular DNA molecules (less than 1pm in length) in mouse B16 cells, whereas TPA increased the number of larger circular DNAs (more than 1 pm in length). Changes in quality and size of circular DNAs were detected by microscale electron microscopy of mica-press adsorption (Yamagishi et al., 1982,1984). Genetic information contained in circular DNAs is being investigated.

D. MITOGENIC EFFECTS Collins and Rozengurt (1982) found that teleocidin was a potent mitogen for murine fibroblasts and that it showed synergistic effects with insulin or with epidermal growth factor (EGF), but not with PDBu. Teleocidin did not stimulate mitogenesis in “TPA non-proliferation 3T3 variants” that retain phorbol ester receptors (Henchman, 1983). Novogrodsky et al., (1984) reported that teleocidin and dihydroteleocidin B were mitogenic to human lymphocytes. Catalase enhanced the mitogenicities of all these teleocidins. All teleocidins were comitogenic to murine peanut agglutinin (PNA)-negative thymocytes treated with phytohemagglutinin, and they induced production of interleukin-2 in these cells. Until recently, it has been impossible to grow normal melanocytes in uitro. However, Eisinger et al. (1983) found that when primary cultures of human epidermis are exposed to TPA the keratinocyte population is selectively killed with outgrowth of normal melanocytes, and the also found that teleocidin and aplysiatoxin, like TPA, are potent enhancers of the growth of human melanocytes. Teleocidin and TPA enhanced cell proliferation of sheep erythrocyte-rosette lymphocytes and induced a high density of cell surface receptors for interleukin-2 and transferrin (Isakov et al., 1985). Dr. N. Shimizu’s group, in collaboration with us, isolated mouse 3T3L1 fibroblast variants, which are unable to respond to the mitogenic stimulation of dihydroteleocidin B. Nonresponsiveness to dihydroteleocidin B is caused by several different lesions, including defects in receptors for insulin or EGF, and in the postreceptor mechanisms (Shimizu et al., 1986). E. OXIDATIVE BURST Troll and associates (1982) found that teleocidin stimulated superoxide anion radical (0;) production by human polymorphonuclear

248

HIROTA FUJIKI AND TAKASHI SUGIMURA

leukocytes and was a slightly stronger stimulator of 0;production than TPA. Similarly, teleocidin had a stronger effect than TPA on nitroblue tetrazolium (NBT) reduction of mouse peritoneal macrophages (Ohkawa et al., 1984)and on activation of the oxidative burst of macrophages (Keisari et al., 1984). In addition, debromoaplysiatoxin and aplysiatoxin enhanced NBT reduction activity (Ohkawa et al., 1985).

F. STIMULATION OF ARACHIDONIC ACID METABOLISM Teleocidin and aplysiatoxin, like TPA, induced the release of arachidonic acid and synthesis of prostaglandins El and F2a in C3H 10T1/2 cells (Umezawa et al., 1981; Horowitz et al., 1983).Teleocidin stimulated prostaglandin release and choline turnover in HeLa cells (Sakamoto et al., 1981), increase in choline release in C3H 10T1/2 cells (Fisher et al., 1982), and synthesis of prostaglandin 12, measured as its nonenzymatic hydrolytic product, 6-ketoprostaglandin Fl,, in a normal rat liver cell line (Snoek and Levine, 1983).

G . EFFECTON MICROFILAMENTS Teleocidin and TPA rapidly stimulated concanavalin A-induced cap formation in mouse T lymphocytes, which was supposed to be due to facilitation of a Ca2+-dependentprocess involving the submembrane actin filaments (Cherif-Zahar et al., 1983). Rifiin et al. (1979) reported that TPA induced loss of the ordered arrangement of microfilaments of chick embryo fibroblasts. In collaboration with Drs. H. Esumi, J. J.-C Lin, and J. R. Feramisco we also observed similar rapid alteration of cytoskeletal organization by teleocidin in rat embryo fibroblasts (REF-52) (Fujiki and Sugimura, 1983). Sakiyama and Hiwasa (1984) reported that teleocidin, dihydroteleocidin B, debromoaplysiatoxin, and TPA induced disruption of microfilaments in BALB/c 3T3 fibroblasts, detected by indirect immunofluorescence microscopy using anti-actin antibodies. An increase in the size of the centrosome, a microtubule-organizing center, in HeLa and human melanoma cells was observed after treatment of the cells with dihydroteleocidin B and TPA (Mascardo and Sherline, 1984).

H. APPEARANCEOF NEW PROTEINS Several groups have analyzed the proteins synthesized in tumor promoter-treated and untreated cells. Teleocidin and TPA caused an increase in the synthesis of two polypeptides with molecular weights

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of 44,000 (p44) and 55,000 (p55). p55 is a component of the cytoskeleton (Bazill et al., 1984). The synthesis of a protein with a molecular weight of 32,000 (p32) in BALB/c 3T3 cells was observed on treatment of the cells with teleocidin, aplysiatoxin, debromoaplysiatoxin, and TPA. Higher concentrations of aplysiatoxin and debromoaplysiatoxin than of teleocidin and TPA were required for the same effect (Hiwasa et al., 1982, 1983). A protein with a molecular weight of 46,000 (p46) was secreted from cultured human hepatoma cells HUH-6 C1-5 into the medium on treatment of the cells with teleocidin, aplysiatoxin, debromoaplysiatoxin, and TPA (Yoneda et al., 1985). EFFECTS I. MISCELLANEOUS Teleocidin, like TPA, elicits the following variety of biochemical and biological responses: inhibition of differentiation of Friend erythroleukemia cells (Fujiki et al., 1979, 1981), irreversible aggregation of human platelets (Kume et al., 198l), induction of histidine decarboxylase activity in mouse skin (Watanabe et al., 1982), inhibition of metabolic cooperation between 6-thioguanine-resistant and 6thioguanine-sensitive cells in a Chinese hamster V79 cell system (Jone et al., 1982), production of large amounts of y-interferon and significant amounts of an interferon-like substance (Wilkinson and Morris, 1983), inhibition of the symbiotic complex formation, pseudoemperipolesis with thymic epithelial cells (Kaneshima et al., 1983), and insulin secretion from isolated pancreatic islets (Yamamoto et al., 1983), decrease in fluorescence anisotropy of C3H 10T1/2 cells (Tran et al., 1983), reduction of the natural killer (NK) cell susceptibility of two established human NK target cell lines, U937 and K562 (Kabelitz, 1984), suppression of cytotoxic T cell development in murine lymphocytes (Yamashita, 1985), induction of ouabain-insensitive DNA synthesis in C3H 10T1/2 fibroblasts (Leister et al., 1985), and reversible depigmentation of mouse melanoma cells by TPA and dihydroteleocidin B (Saeki et al., 1985).

MORE STRONGLY BY TELEOCIDIN THAN BY TPA J. EFFECTSINDUCED Beside the various biological responses that are induced in a similar way and with the same specific activities by teleocidin, aplysiatoxin, and TPA, two remarkable effects were found to be induced 100 times more effectively by teleocidin than by TPA. First, when malignant transformation of A31-1-1 mouse cells that were treated with 3-methylcholanthrene and subsequently incubated with either teleocidin or

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TPA was assayed by scoring transformation foci, dihydroteleocidin B was found to be at least 100 times more effective than TPA in enhancing transformation (Hirakawa et al., 1982). Second, Borek and associates (1984) reported that teleocidin was over 100 times more effective than TPA in enhancing radiogenic transformation of C3H 10T1/2 cells when thyroid hormone was added to the medium. Whether these differences in potencies of the two tumor promoters are related to structural differences or to differences in stability of the compounds remains to be elucidated.

K. INTERTISSUE AND INTERSPECIES VARIATION The tumor-promoting activity of croton oil or TPA is species specific for mouse skin (Berenblum, 1978). The important question of whether TPA-type tumor promoters have a role in promotion in human carcinogenesis has not yet been answered. Chida and Kuroki (1983) demonstrated that intact human epidermal keratinocytes and dermal fibroblasts have specific 3H-PDBu binding sites and that the specific 3H-PDBu binding is inhibited by teleocidin. However, they found that there was no down regulation of the binding sites in human epidermal cells. They also showed that teleocidin, with TPA, inhibits DNA synthesis and uptake of 2-deoxy-~-glucosebut does not induce ODC activity in human epidermal cells. Therefore, the effects of TPAtype tumor promoters on human epidermal cells and mouse epidermal cells seem to be different (Chida and Kuroki, 1984). In contrast with the above-mentioned results, Verma et al. (1985) recently found that human skin, like mouse skin, responded in human skin punch biopsy samples to TPA for in vitro induction of ODC activity. It appears that the effect of TPA on ODC induction in intact human skin is different from that in cultured human epidermal cells (Verma et al., 1985). Improvement of culture techniques has made it possible to investigate the action of tumor promoters on normal human bronchial epithelial (NHBE) cells. Harris and associates studied intertissue differences in the effects of tumor promoters. Teleocidin and TPA increased the ODC activity of NHBE cells in maintenance medium without EGF but decreased the ODC activity in a growth medium containing EGF (Willey et al., 198413).However, teleocidin and TPA increased plasminogen activator activity and decreased aryl hydrocarbon hydroxylase activity in both media. Similar results were obtained with aplysiatoxin and debromoaplysiatoxin (Willey et al., 1984a). Willey et al. concluded that the changes induced by TPA-type tumor

NEW CLASSES OF TUMOR PROMOTERS

25 1

promoters are consistent with induction of terminal squamous differentiation of normal, uninitiated cells, while initiated cells may be resistant to induction of this differentiation (Hawley-Nelson et al., 1982). VI. Mechanism of Tumor Promotion by TPA-Type Tumor Promoters

A. ACTIVATIONOF PROTEIN KINASE C The three classes of tumor promoters, phorbol ester, teleocidin, and aplysiatoxin, are TPA-type tumor promoters, because they bind to the same receptors as TPA on cell membranes. Recently, Nishizuka (1984) reported that Ca2+-activated,phospholipid-dependent protein kinase (protein kinase C) serves as a receptor for phorbol ester and that TPA activates phosphorylation of H 1histone by partially purified protein kinase C in uitro. Protein kinase C is activated by diacylglycerol, which is normally almost completely absent from membranes but is transiently produced from inositol phospholipids in response to extracellular signals. In collaboration with Dr. Y. Nishizuka’s group we demonstrated that teleocidin and debromoaplysiatoxin, like TPA, directly activate phosphorylation of H1 histone by protein kinase C obtained from rat brain (Fujiki et al., 1984e). The dose-response curves for teleocidin and debromoaplysiatoxin are compared with that for TPA in Fig. 8. I

7

z

2

X

E

8

1

.-2. .->

5 m

1

W

m 3 .-C W c

e

n

0

-/I

I

I

I

10

100

1 000

Concentration ( ng/ml )

FIG.8. Dose-dependent activations of protein kinase C from the soluble fraction of rat brain by TPA (0), teleocidin (O),and debromoaplysiatoxin (A).

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The concentrations needed for half the maximum activation of protein kinase C were approximately 3 ng/ml for TPA, 40 ng/ml for teleocidin, and 400 ng/ml for debromoaplysiatoxin. This difference might be due to the different lipophilicities of these three classes of tumor promoters. We extended our study to include activation of bovine brain protein kinase C by dihydroteleocidin B, lyngbyatoxin A, two teleocidin A isomers, four teleocidin B isomers, indolactam-Vs, olivoretins, and several aplysiatoxin derivatives (Fujiki et al., 1986~). These studies showed that the presence of tumor-promoting activities of these compounds was consistent with their activations of protein kinase C. Teleocidin and aplysiatoxin were also shown to activate bovine brain protein kinase C (Arcoleo and Weinstein, 1985). These findings support our idea that teleocidin and aplysiatoxin cause an initial reaction by the same process of phosphorylation as that with TPA. The physiological substrate(s) of protein kinase C in mouse skin is unknown. Cochet et al. (1984) examined the EGF receptor as a possible membrane substrate for protein kinase C, since teleocidin, aplysiatoxin, and TPA inhibited the specific binding of EGF to its cellsurface receptor (Lee and Weinstein, 1978; Shoyab et al., 1979; Umezawa et al., 1981; Horowitz et al., 1983; Shimomura et al., 1983). They found that protein kinase C phosphorylates threonine residues, in both purified EGF receptor and membrane-associated EGF receptor, and they obtained the same three major phosphothreonine-containing peptides from both (Cochet et al., 1984). The EGF receptor protein, which possesses tyrosine protein kinase activity, is self-phosphorylated in the presence of EGF (Cohen et al., 1982). Rosner and associates showed that teleocidin, aplysiatoxin, and debromoaplysiatoxin, like TPA, inhibit EGF-stimulated phosphorylation of tyrosine residues of the receptor in A431 cells (Friedman et al., 1984). These results can be summarized as follows: the tumor promoter activates protein kinase C, which catalyzes phosphorylation of threonine residues of the EGF receptor on A431 cells, and this initial reaction causes both inhibition of phosphorylation of tyrosine residues of the EGF receptor and loss of EGF binding to the apparent high-affinity EGF receptor.

B. EXPRESSION OF ONCOGENES It is worthwhile to discuss briefly the effects of tumor promoters in modulation of oncogene expression, because various activated oncogenes have been found in human tumors and tumor cell lines by DNA transfection with the NIH 3T3 cell line. TPA treatment stimulated the

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expression of c-myc mRNA and c-fos mRNA in BALB/c 3T3 cells (Kelly et al., 1983; Greenberg and Ziff, 1984) and of the c-fos gene during differentiation of a monocytic cell line, U-937, and HL-60 cells (Mitchell et al., 1985), whereas expression of c-myb in human myeloblastic leukemia (ML-1) cells decreased during differentiation induced by TPA (Craig and Bloch, 1984).An activated c-rasHgene was demonstrated in papillomas induced in two different mouse strains by initiation with DMBA followed by multiple applications of TPA, whereas DNA from epidermis treated once with TPA did not induce any transformants (Balmain et al., 1984). Recently, the levels of expression of the protooncogenes rmH,rmK,fos, myc, abl, and raf at different stages of tumor development in mouse skin after treatment with TPA were determined. No significant increases in expression of these protooncogenes were found. A reduced level of expression of abl was observed in tumors and after repeated treatments with TPA (Toftgard et al., 1985). The effects of tumor promoters on the process of DNA transfection were also studied in C3H 10T1/2 cells and the NIH 3T3 cell line. Teleocidin and TPA caused about 5-fold increase in the number of transformed foci obtained in C3H 10T1/2 cells transfected with the T24 oncogene (Hsiao et al., 1984), a human bladder carcinoma oncogene. Weinstein and associates reported that teleocidin has an even greater effect than TPA when calculated on a molar basis. On the contrary, the number of transformed foci obtained in the NIH 3T3 cell line decreased after similar treatment with either teleocidin or TPA. Teleocidin or TPA appears to complement the function of activated cellular oncogenes (Hsiao et al., 1984). This idea is supported by the following experiment. Connan et al. (1985)transferred genes encoding the large-T protein of polyoma virus (plt),adenovirus E1A genes, the viral myc gene (v-myc), or rearranged forms of the cellular c-myc gene into rat embryo fibroblasts (REFs) or established FR3T3 rat cells by the protoplast fusion technique. Transformed foci were induced in these plt- and myc-immortalized cell lines by treatment with TPA. These results indicate that tumor promoters allow transformation of cells that have previously been either initiated by carcinogens or immortalized by activated oncogenes. Recently Dotto et al. (1985) showed that in the presence of tumor promoters, ras-containing primary REFs acquire the ability to overgrow normal cells in the monolayer and to form foci with 100% efficiency. Further studies with tumor promoters are necessary to establish the connection between the above evidence and tumor formation in the cells of mouse skin.

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VII. Non-TPA-Type Tumor Promoter, Palytoxin

Studies on TPA-type tumor promoters raised the questions of whether the newly found tumor promoters are TPA-type and whether compounds that give positive results in the irritant test but negative results in tests on induction of ODC or induction of HL-60 cell adhesion are tumor promoters in mouse skin. We examined palytoxin, which is a very strong irritant of mouse ear but does not induce ODC activity in mouse skin or adhesion of HL-60 cells (Fujiki et al., 1984d). Palytoxin with a molecular weight of 2,681 is a powerful water-soluble toxin isolated from a marine coelenterate of the genus Palythoa. Figure 9 shows the structure of palytoxin, determined independently by Moore in Hawaii (Moore and Bartolini, 1981) and Hirata’s group in Japan (Uemura et al., 1981). The Hawaiian word lirnu-make-o-Hana, which means “the deadly seeweed of Hana,” clearly describes this toxic substance, later identified as “palytoxin.” Palytoxin has for long been veiled in myth and legend. Several biological activities of palytoxin are summarized in the following paragraph to facilitate its comparison with those of the TPAtype tumor promoters teleocidin, aplysiatoxin, and TPA (described in Sections III,A, III,B, and IV,A). The ID;! value of palytoxin in the irritant test was 0.060 nmol/ear, which corresponds to only 0.2 pg, indicating that it is a very strong irritant. Palytoxin was so toxic that mice died within 4 hr after application of either 33 or 165 pg per

OH

OH FIG.9. Structure of palytoxin.

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mouse. No induction of ODC activity was observed 4 hr after application of palytoxin at doses of 0.3-8.25 pg. Palytoxin at concentrations of 0.04-0.2 ng/ml did not induce adhesion of HL-60 cells. Furthermore, at concentrations of up to 10 p M , it did not inhibit the specific binding of 3H-labeled TPA to a mouse particulate fraction. Trosko and Moul6 independently found that palytoxin did not inhibit metabolic cooperation (Trosko and Moul6, unpublished results). Of the various biological activities that are strongly induced by TPA-type tumor promoters, palytoxin induced only irritation of mouse ear. It was further tested in a two-stage carcinogenesis experiment on mouse skin. In this experiment, from 1 week after initiation 0.5 pg of palytoxin was applied to the skin twice a week. Only about 60% of the mice in the groups treated with DMBA plus palytoxin or with palytoxin alone survived until week 30, because of its strong toxicity. The percentage of tumor-bearing mice in the group treated with DMBA plus palytoxin was 62.5 in week 30, and the average number of tumors per mouse was 1.0. No tumors were observed in groups treated with DMBA or palytoxin alone (Fujiki et al., 1986b). In collaboration with Dr. K. Ohuchi’s group, we reported that when various concentrations of palytoxin and TPA were incubated with rat macrophages they released prostaglandin Ez. It was found that palytoxin caused prostaglandin Ez release at a rate 300 times that of TPA (Ohuchi et al., 1985). Dr. L. Levine in collaboration with us confirmed that palytoxin was 1,000 to 3,000 times more effective than TPA-type tumor promoters on the stimulation of arachidonic acid metabolism using rat liver cells in culture (Levine and Fujiki, 1985). Furthermore, the stimulation of arachidonic acid metabolism was found to be synergistic in the presence of palytoxin with EGF, transforming growth factor-a, or -/3 (Levine et al., 1986). During our research, we found that thapsigargin is also a non-TPAtype tumor promoter in mouse skin. Thapsigargin, a sesquiterpene lactone, is the major skin irritating constituent of the roots of Thapsia garganica L. (Umbelliferae) and has histamine-liberating activity. Christensen et al. (1980) identified it as a hexaoxygenated Cs guaianolide esterified with four carboxylic acids (Fig. 10). In collaboration with Dr. S. B. Christensen, we found that thapsigargin induces irritation of mouse ear but not ODC activity or adhesion of HL-60 cells. Like palytoxin, thapsigargin did not inhibit the specific binding of 3Hlabeled TPA. However, of mice treated with DMBA plus thapsigargin tumors were found in 53.5% in week 30 (average number, 0.7 per mouse) (Hakii et al., 1986). The non-TPA-type tumor promoters palytoxin and thapsigargin

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HIROTA FUJIKI A N D TAKASHI SUGIMURA

FIG.10. Structure of thapsigargin.

have the common biological activities of skin irritation and promotion of carcinogenesis in mouse skin. Furthermore, both compounds did not induce ODC activity in mouse skin four hours after their application or adhesion of HL-60 cells. The absence of receptor binding by palytoxin and thapsigargin is compatible with our finding that neither of these two compounds activated protein kinase C in vitro (Fujiki et aZ., 1986b). Thus these two non-TPA-type tumor promoters presumably do not exert their biological activities, including tumor promoting activity, through phorbol ester receptors on the cell membranes. Moreover, they may not act through the same pathway, since thapsigargin induces histidine decarboxylase activity in mouse skin but pa1986).Studies on lytoxin does not (Watanabe et d.,1982; Hakii et d., broader aspects of the effects of non-TPA-type tumor promoters may be helpful in solving the problems of interspecies and intertissue variations in responses to tumor promoters. We have recently demon-

TPA-type tumor promoter

0 / , '

Common biological and biochemical effects

Non-TPA-type tumor promoter \Membrane

F ~ G11. . Mechanisms of tumor promotion by TPA-type and non-TPA-type tumor promoters.

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strated that the TPA-type and the non-TPA-type tumor promoters commonly induce several biological effects such as irritation of mouse ear, stimulations of prostaglandin Ez production and of arachidonic acid metabolism, and stimulation of superoxide anion production, in addition to tumor promoting activity. These common effects seem to be the most essential biological activities for tumor production (Fig.

11). VIII. Conclusion

Since the discovery that, like TPA, dihydroteleocidin B is a potent tumor promoter, we have extended studies on two classes of TPA-type tumor promoters, teleocidin and aplysiatoxin, and on the non-TPAtype tumor promoters palytoxin and thapsigargin. These TPA-type tumor promoters have provided several important new pieces of evidence, most of which are summarized in this review. In contrast to phorbol esters, which are present only in plants, such as the families Euphorbiaceae and Thymelaeaceae, the new tumor promoters described in this review have been found in a wide range of living things, including Streptomyces, Streptouerticillium, bluegreen algae, coelenterates, and umbelliferous plants. Epidemiological studies have not been conducted on the relation between human exposure to these compounds and the incidence of human skin cancer, but attention has been attracted to these new tumor promoters by their irritation of human skin, such as in swimmer’s itch in Hawaii and Japan. Since teleocidin A-1 is present in both Streptomyces and bluegreen algae, it may be synthesized by algae, Streptomyces, Streptouerticillium, and other fungi. An interesting finding was that a biosynthetic intermediate of teleocidins, (-)-indolactam-V, has weak tumor-promoting activity but not strong activity in the irritant test. Other Streptomyces and blue-green algae biosynthesizing (-)-indolactam-V may be present in our environment. Because TPA has been shown to induce hyperplasia in human skin grafted onto mice (Yuspa, 1979) as well as to induce ODC activity in human skin punch biopsy samples (Verma et aZ., 1985), it would be worthwhile to investigate the distributions of weak tumor promoters such as these from the standpoint of human carcinogenesis. From the time of the first demonstration of two-stage chemical carcinogenesis, tumor promotion has been associated with inflammation. The new TPA-type and non-TPA-type tumor promoters described in this article are all irritants to mouse ear, causing inflammation of the skin. Further studies are necessary on whether the effects of these

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tumor promoters are related to chronic inflammation in human tissue. We still do not know the cause of human cancer, but the process of inflammation associated with cell proliferation may contribute to tumor promotion in human tissues. Teleocidin, aplysiatoxin, and also non-TPA-type tumor promoters should be useful in investigating the heterogeneous steps in the complicated process of promotion at the biochemical and molecular levels. Such studies on tumor promotion should achieve an understanding of tumor promotion in human cancer development. ACKNOWLEDGMENTS This work was supported in part by grants-in-aid for Cancer Research from the Ministry of Education, Science and Culture, Overseas Scientific Research Survey (Cancer Program), and a Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan, and by grants from the Foundation for Promotion of Cancer Research, the Princess Takamatsu Cancer Research Fund, the Japanese Foundation for Multidisciplinary Treatment of Cancer, and the Adult Disease Clinic Memorial Foundation of Japan. Work on the isolation of lyngbyatoxin A and aplysiatoxin derivatives used in this study was supported by Grant CA 12623-11 to Dr. R. E. Moore, University of Hawaii, from the National Cancer Institute, Department of Health and Human Services. We would like to express our sincere appreciation to Dr. M. Suganuma, Mrs. M. Nakayasu, and other colleagues in our Institute and to many Japanese scientists including Drs. K. Shudo, S. Sakai, H. Nishino, R. Kato, T. Kakunaga, Y. and N. Shimizu, Y. Hirata, and D. Ueniura and many overseas scientists including Drs. R. E. Moore, I. B. Weinstein, E. Hecker, L. Levine, T. J. Slaga, W. Troll, J. E. Trosko, E. Huberman, G. Klein, L. Eliasson, C. Borek, A. R. Kinsella, M. R. Rosner, A. Novogrodsky, and S. B. Christensen for their collaborations.

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ANTlCARClNOGENIC ACTION 0 F P ROTEASE INHI BIT0RS Walter Troll, Rakorna Wiesner, and Krystyna Frenkel Department Of EnvironmentalMedicine, New York University Medical Center, New York, New York 1W16

I. Introduction

A possible approach to devising ways of controlling cancer in man is to introduce changes in nutrition. Epidemiological studies have shown that cancers that are not caused by cigarette smoking (breast, colon, and prostatic) are directly related to meat/fat consumption and may be prevented by the ingestion of vegetables. A number of compounds occurring in vegetables have been identified as cancer preventive or “chemopreventive” agents. The first group of agents identified as specific inhibitors of tumor promotion were protease inhibitors (Troll et al., 1970). Other groups included vitamins, hormones, and antioxidants (Wattenberg, 1985). The discovery of the existence of chemopreventive agents, agents that block the multistage events of carcinogenesis, has provided new opportunities to study the mechanism(s) of tumor formation. Protease inhibitors, when incorporated into the diet, were shown to suppress cancer in experimental animal models including skin (Troll et al., 1979), breast (Troll et al., 1980), colon (Weed et al., 1985), and liver cancers (Becker, et al., 1981). However, their use as chemopreventive agents in man has as yet not been considered. A major difficulty in introducing any chemopreventive agent for use by the general population is the requirement for negligible toxicity. The use of vitamins as chemopreventive agents was proposed because information on their nutritional role in humans was available. Vitamins have enjoyed advance positive publicity based on their presumptive nutritional soundness. The most promising vitamins were A and C which, however, were given at higher doses as chemopreventive agents than when used as dietary supplements (Loprinzi and Verma, 1985). On the other hand, the role of protease inhibitors in the diet has been viewed with suspicion because they seem to limit the growth of young animals (Liener and Kakade, 1980).Thus, in compari265 ADVANCES IN CANCER RESEARCH, VOL 49

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$on with vitamins, protease inhibitors like Cinderella start with the distinct disadvantage of being an unnecessary additive to the diet of the growing animal. This article will show that protease inhibitois in the diet prevent cancer by interfering with cancer development b y a variety of mechanisms. Protease inhibitors occur naturally in plants and inay have evolved as a defense mechanism of plants toward invading insects (Birk 1974; Richardson, 1977). It was shown that insects cause the induction of‘protease inhibitors in plants (Ryan, 1980),which seems to confirm their presumptive rcttc of fending off destruction by insects. Protease inhibitors seem to act b y inhibiting the insects’ digestive enzymes and, thus, preventing their digesting the stems and leaves of plants. In man, protease inhibitors prevent the action of trypsin and chymotrypqin (Birk, 1976). This action b y the protease inhibitors counteracts the development of tuniors by limiting the overproduction of amino acids that are needed more by the growing tumor than by normal tissue.

11. Role of Plasma Protease Inhibitors in Disease

Protease inhibitors also occur in substantial concentrations in animals and in man. For example, protease inhibitors, such as az-macroglobiilin, cr,-protease inhibitor, and inter al-trypsin inhibitor, are major constituents in human plasma amounting to about 10% of the total proteins. There are several examples which show that the congenital absence of these inhibitors leads to disease in humans. Thus, the lack ofcr,-trypsin inhibitor formation in certain f h i l i e s leads to an early onset of emphysema (Travis and Salvenson, 1983). That this inhibitor is directly involved in preventing emphysema is confirmed b y the observation that its destruction in cigarette smokers, through the oxidation of an essential inethionine of the inhibitor by activated inflammatory cells, led to emphysema (Cai-p and Janoff, 1978; Janoff et nl., 1979).The contribution of the destruction of this inhibitor to lung cancer remains to be established. The direct relation of protease inhibitors to cancer has been shown in the case of two other plasma protease inhibitors. T h e inter a,-trypsin inhibitor is destroyed in 3060% of individuals with disseminated neoplastic disease, which results in the urinary excretion of an immunologically recognizable glycoprotein portion derived from the inhibitor (Chawla et uZ., 1978). The major protease inhibitor, 01-macroglobulin inhibitor, is totally absent in neoplastic lesions (Saksela, 1985). Some plasma protease inhibitors control the functions of biologi-

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cally important proteases such as thrombin, plasminogen activator, plasmin, and protease involved in complement activation (Travis and Salvesen, 1983).These and other proteases have been shown to contribute to the metastatic potential of cancer cells (Liotta, 1986). Protease inhibitors, capable of inhibiting collagenase or of blocking the protease cascade which leads to its activation, were shown to block the metastatic progress (Rifkin and Crowe, 1977; Roughly et aZ., 1978). Other protease inhibitors, such as the inhibitor of plasminogen activator, are also capable of blocking the metastatic process, since it has been shown that antibodies to plasminogen activator inhibit tumor metastases (Ossowski and Reich, 1983). Thus, protease inhibitors present in human plasma may serve at least in part to prevent cancer formation and its metastases. The stimulation of synthesis of proteases required for metastases has been reported to be a function of oncogene expression (Liotta, 1986). It is of interest that expression of the myc oncogene in C3H 10TV2 cells has been shown to be inhibited by a series of protease inhibitors, such as antipain (Chang et al., 1985) and Bowman-Birk inhibitor (BBI) (A. R. Kennedy, personal communication), which also are known to prevent neoplastic transformation in the same cells (Kennedy and Little, 1981; Borek et al., 1979; Kuroki and Drevon, 1979). Transfection of ras oncogene to NIH 3T3 cells was suppressed by leupeptin, antipain, anti crl-trypsin, and .z-aminocaproicacid (Garte et al., 1987). Thus, chemoprevention mediated by protease inhibitors might proceed by a variety of mechanisms that inchde blocking tumor promotion, oncogene expression, and metastatic progression. Human epidemiological studies have shown that consumption of seeds containing protease inhibitors in high concentrations significantly lowers the occurrence of breast, colon, and prostatic cancers (Correa, 1981). That protease inhibitors are the putative agents in this chemopreventive action has been corroborated by the results of animal experiments in which a variety of protease inhibitors that were included in the diet were shown to suppress skin (Troll et al., 1979), breast (Troll et al., 1980), colon (Weed et al., 1985), and liver cancers (Becker, 1981). Protease inhibitors have also been shown to suppress neoplastic transformation in vitro (Kennedy and Little, 1981; Borek et al., 1979; Kuroki and Drevon, 1979; Yavelow et al., 1985).The mechanism for this action is not known but appears to involve multiple processes including suppression of oxygen radical formation (Goldstein et al., 1979), induction of poly(ADP-ribose) (Cleaver et al., 1986), inhibition of selective DNA amplification (Heilbronn et al., 1985), and prevention of oncogene expression (Chang et al., 1985; Garte et al., 1987).

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While there may be considerable support for the view that protease inhibitors play a role in the prevention or in the moderation of cancer progression (expression), recommending an increase in the consumption of protease inhibitors in order to prevent cancer remains an uphill task. The precise daily requirements for a cancer preventive agent presents technical difficulties exceeding those for staple nutrients such as proteins and vitamins. While the usefulness of cancer preventive agents is clearly established from epidemiological studies and animal models, development of standards for requirements in miin will require new techniques. A possibly useful method for futiire experiments comes from recent observations that protease inhibitors are capable of selective prevention of DNA expression of the inyc gene (Chang et aZ., 1985) and of DNA polymerase a (Heilbronn et al., 1985). ‘The effect of plasma supplemented with appropriate protease inhibitors on these particular processes may offer the opportunity of setting standards for the use of cancer preventive protease inhibitors.

111. Inhibition of Carcinogenesis by Protease Inhibitors

A. INHIRITION OF SKIN CANCER BY

I)IRECT

APPLICATIONTO

SKIN

Chemical carciriogenesis is a multistep process. Two major stages have been identified in mouse skin carcinogenesis: (1) initiation achieved by a single subthreshold dose of n carcinogen and (2) promotion by a noncarcinogenic inflamniatory agent (Bercnblum, 1941; Hecker, 1968; Boutwell, 1964). The most studied promoter has been the active inflammatory component of croton oil, phorl~ol-12-myristate-13-acetate (PMA), identified as such and isolated by Van Duuren and Orris (1965) and by Hecker (1968). Application of PMA to skin results in inflammation which causes an infiltration of neutrophils (Janoff et al., 1970).Chemotactic factors released by neutrophils lead to further infiltration of these cells. Stimulation of neutrophils by PMA results in the production of active oxygen species (Goldstein et al., 1979) that are capable of causing multiple types of biological damage including DNA breakage and modification (Birnboim, 1982a,b, 1983; Birnboim and Biggar, 1982; Emerit and Cerutti, 1981a; Troll et al., 1984; Frenkel et al., 1986b,c). In addition, PMA induces two types of enzymes in a number of biological systems: (1) a specific protein, protein kinase C, which appears to be a specific receptor for PMA (Ashendel et al., 1983; Vandenbark and Niedel, 1984), and (2) a number of proteases, including plasminogen activator and other trypsin-

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like proteases, that are capable of hydrolyzing tosyl-L-arginine methyl ester (TAME) (Troll et al., 1954) and protamine (Troll et al., 1975). To test for a possible role for proteases in promotion, a variety of protease inhibitors were applied to the skin of mice 7 hr before the promoter. Significant inhibition of tumor promotion occurred when inhibitors of trypsin and chymotrypsin were used (Troll et al., 1970; Hozumi et al., 1972). Two of the inhibitors tested, tosyl-L-lysine chloromethyl ketone (TLCK) and tosylphenylalanine chloromethyl ketone (TPCK), were shown not only to block tumor promotion but also to inhibit rat brain protein kinase C, the receptor for PMA (Solomon et

al., 1985). B. PREVENTION OF CANCER BY PROTEASE INHIBITORS IN THE DIET

1. Leupeptin, Antipain, and Chymostatin Feeding protease inhibitors to experimental animals in which cancer is induced has demonstrated that a diet containing appropriate protease inhibitors is capable of suppressing cancer. Leupeptin [acetyl-(propy1)leucine-leucinearginall was the first naturally occurring protease inhibitor described that had wide effectiveness as an anticarcinogen (Matsushima et al., 1975). Leupeptin belongs to a family of small protease inhibitors which was purified from streptomycetes by Umezawa (1972). Leupeptin, antipain, and chymostatin are peptides containing an aldehyde instead of a carboxyl group in the terminal amino acid. It is this terminal amino aldehyde that determines the type of protease that is inhibited. Reduction of the carboxyl group of the terminal arginine in both leupeptin and antipain to form “arginal” results in the inhibition of the trypsin family of enzymes including plasminogen activator. Chymostatin, which has the carboxyl of the terminal phenylalanine reduced to an aldehyde, inhibits chymotrypsin. These three protease inhibitors in addition to inhibiting specific serine enzymes such as trypsin and chymotrypsin also inhibit the sulfhydryl proteases papain, ficin, and cathepsin (Umezawa, 1972). The incorporation of leupeptin into the diets of mice and rats suppressed the induction of tumors in mouse skin, rat colon, and esophagus as well as leukemia (Matsushima et al., 1975). The diet appeared to be nontoxic and had no significant effect on the weight of the animals. The feeding of leupeptin was the first demonstration that incorporation of a nontoxic protease inhibitor in the diet suppressed a wide range of experimental tumors. The disadvantage of leupeptin as an ideal preventive agent is that it is extensively metabolized, which

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leads to the release of the amino acid leucine and its increased concentration in the urine (Tanaka, 1983). Interestingly, this increased concentration of leucine acts as a tumor promoter in experimentally induced bladder cancer (Kakizoe et al., 1982, 1983; Nishio et d . , 1986). Although leucine is essential in nutrition, an excess of this amino acid appears to benefit the growing cancer cell. That amino acids are indeed required for the growth of cancer cells was shown by the inhibition of the growth of the mouse hepatoma BW-7756 and the mouse manimary adenocarcinonia CjHBA when limited amounts of phenylalanine and tyrosine were present in the diet (Sngimtira et al., 1959; Loriiicz et ul., 1969). Furtherinore, a clinical report showed a remission of abdominal tumors in a 54-year-old woman with iidenocarcinoma of the ovary and abdominal metastasis, when she was put on a diet with lowered amounts of phenylalanine and tyrosine (Lingeman, 1974). Metastasis of Lewis lung carcinoma was inhibited by a leucine-restricted diet (Chan ef d . , 1983). Conversely, an excess of leucine due to the metabolism of leupeptin caused a more rapid growth of induced bladder cancer in rats and, thus, confirmed the potential of specific amino acids to enhance growth of tumors. Therefore, the release of leucine limits the use of leupeptin as a cancer preventive agent (Kakizoe et ul., 1984). Restriction of amino acid availability can be accomplished by inclusion of natural protease inhibitors in the diet, which would decrease digestion of proteins, and conseqnently would lower the yield of amino acids (Yavelow et al., 1983).

2 . Protease lnlzibitors from Soyheuns Consumption by rodents of raw soybeans or of purified fractions from soybeans that contain protease inhibitors has been shown to suppress skin (Troll et al., 1979), breast (Troll et al., 1980), colon (Weed et ul., 1985)and liver cancers (Becker, 1981). Skin tumors induced in mice by 4-nitroquinoline N-oxide followed by PMA treatment were suppressed when casein in the diet was replaced b y raw soybeans with an equivalent protein content. Destruction ofthe proteaoe inhibitors by heating the raw soybeans abolished their ability to suppress promotion when they were fed to mice (Troll et al., 1979). Moreover, a diet containing raw soybeans suppressed breast cancer caused by ionizing radiation in Sprague-Dawley rats (Troll et al., 1980). Purified fractions from soybeans which contain protease inhibitors suppressed spontaneous liver cancer in the C3H HeN mice (Becker, 1981). A fraction enriched in BBI blocked l,2-dimethylhydrazine-induced colon cancer in mice (Weed et ul., 1985).

ANTICARCINOGENIC ACTION OF PROTEASE INHIBITORS

27 1

Protease inhibitors ingested by eating seeds or legumes may inactivate the proteases trypsin and chymotrypsin in the duodenum, as has been shown to occur in mice and rats fed BBI. 1251-LabeledBBI fed to animals was found mainly in the intestinal tract and feces. HPLC analysis of 1251-labeledmaterial isolated from the feces revealed that its molecular weight was 33,000, precisely what was expected for a complex of trypsin or chymotrypsin with BBI (25,000 + 8,000). Corroboration of that composition was obtained when, upon acidification of the complex, free 125-labeledBBI was released (Yavelow et al., 1983). Diets containing protease inhibitors such as Bowman-Birk are capable of diminishing the occurrence of a variety of cancers in organs where the inhibitor is not present. This points to an indirect role that proteases may play in the modulation of carcinogenesis. The proteins that we eat are digested by proteases to peptides and then to amino acids which appear in the blood. If these amino acids are not oxidized or utilized for protein synthesis, they are excreted in the urine. We have already mentioned that the presence of the amino acid leucine in the urine accelerated bladder cancer. A high protein (20% casein) diet started 6 weeks following the initiation of carcinogenesis with aflatoxin B1 caused formation of preneoplastic lesions in the liver, whereas these lesions did not develop in animals fed a low protein (5%)diet. High protein diets given during initiation with aflatoxin had no effect on the yield of preneoplastic lesions (Appleton and Campbell, 1983). These findings are similar to those shown by Tannenbaum and Silverstone (1953), who demonstrated that high-calorie and high-fat diets increased the yield of skin tumors caused by benzo[a]pyrene. Thus, the increased availability of amino acids in populations on a high protein diet may be a factor contributing to breast, colon, prostate, oral, and pharyngeal cancers. Therefore, it is possible that the presence of an excess of amino acids in the nongrowing adult provides a stimulus for transformation and/or growth of tumor cells.

3. Synthetic Protease Inhibitors

The suggestion that incorporation of protease inhibitors in the diet suppresses experimentally induced carcinogenesis was confirmed using a synthetic trypsin inhibitor, N,N-dimethylamino-[p-(p’-guanidinobenzoloxy)]benzilcarbonyloxyglycolate. It suppressed 7,12-dimethylbenz[alanthracene (DMBA)-induced mammary carcinogenesis (Yamamura et al., 1978) in a dose-related manner. [N,N-Dimethylcarbamoylmethyl-4-(4-guanidinobenzoyloxy)phenylacetate]methane sulfate, which inhibits both trypsin and chymotrypsin, prevented skin carcinogenesis induced by 3-methylcholanthrene (Ohkoshi and Fujii,

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1983).The simplest protease inhibitor used in feeding experiments to suppress cancer was E-aminocaproic acid, a known inhibitor of trypsin and plasniinogen activator. It is derived from the amino acid lysine from which the a-amino group is removed. This inhibitor, when given in the drinking water, successfully suppressed colon cancer that was induced by 1,2-dimethylhydrazine in mice (Corasanti et aZ., 1982).

c. INHIBITION OF NEOPLASTICTRANSFORMATION in VitrO Protease inhibitors have been shown to suppress neoplastic transformation in C3H 10TV2 cells (Borek et al., 1979; Kuroki and Drevon, 1979; Kennedy and Little, 1981). In these experiments, 10TV2 cells were initiated with ionizing radiation and promoted b y PMA (Kennedy, 1984).The application of protease inhibitors at different stages permitted differentiation between the protease inhibitors that were most effective in counteracting X-ray transformation or enhancement by PMA. Antipain and chymostatin were effective in suppressing both initiation and promotion. Leupeptin was more effective against X-ray transformation than PMA promotion. BBI inhibited only X-ray transformation, while Kunitz soybean trypsin inhibitor (SBTI) inhibited only PMA promotion. The striking difference in the action ofthe two major soybean protease inhibitors, SBTI and BBI, is of particular interest. The major difference with respect to protease inhibition is that BBI inhibits chymotrypsin to the same extent a s it does trypsin while SBTI primarily inhibits trypsin. BBI is a double-headed inhibitor with one end containing lysine-serine, the trypsin inhibitory site, and the other end containing leucine-serine, the chymotrypsin inhibitory site (Birk, 1976). When the trypsin inhibitory site is destroyed by limited proteolysis, the intact chymotrypsin inhibitory site is still fully active. This modified BBI, which is capable of inhibiting only chymotrypsin, suppresses X-ray transformation of C3H 10TV2 cells even when used in nanogram quantities (Yavelow et al., 1985). tion is not as easily generalized in relation to the protease they inhibit. Antipain and chymostatin inhibit both X-ray transformation and tumor promotion (Kennedy, 1984), yet antipain inhibits only trypsin while chymostatin inhibits only chymotrypsin (Umezawa, 1972). There may be other overriding factors which influence the overall action of protease inhibitors (i.e., the effectiveness of entering cell membranes or receptor interaction). Moreover, inhibitors may act indirectly by affecting other biological parameters, e.g., the formation of oxygen radicals (Goldstein et al., 1979), inhibition of poly(ADP-ribose) (Cleaver

ANTICARCINOGENIC ACTION OF PROTEASE INHIBITORS

273

et al., 1986), or oncogene expression (Chang et al., 1985). Since each of these biological functions may be governed by a different protease, different protease inhibitors may be required to counteract the action of those proteases.

D. OTHERACTIONSOF PROTEASE INHIBITORS Another interesting in vitro action of protease inhibitors, that of the reduction of spontaneous chromosome abnormalities in cells with Bloom’s syndrome, has been noted by Kennedy et al. (1984a). They demonstrated that the frequency of abnormalities in Bloom’s syndrome is lowered by SBTI, BBI, and antipain. Bloom’s syndrome is a genetic disease characterized by a high level of sister chromatid exchanges (SCE) (Chaganti et al., 1974) and by an inadequate handling of oxygen metabolism (Cerutti, 1982). The question of whether the action of protease inhibitors involves a direct inhibition of protease( s) or whether it is due to blocking the formation of oxygen radicals remains to be answered. Free radicals are capable of increasing SCE (MacRae and Stich, 1979). In patients with Bloom’s syndrome excessive amounts of oxygen radicals may be formed (Cerutti, 1982) or a deficiency in detoxication of those radicals may occur (Emerit and Cerutti, 1981a). Protease inhibitors are known to prevent the formation of superoxide anion radicals (0;) and hydrogen peroxide (HzOz) by human polymorphonuclear leukocytes (PMNs) that are activated by tumor promoters or by other stimuli (Goldstein et al., 1979; Frenkel et al., 1986d). It might be that it is the inhibition of chymotrypsin by a variety of inhibitors that is directly related to suppression of HzOz formation by promoter-induced human neutrophils (Frenkel et a1., 1986d). Our interest in chymotrypsin inhibitors started with our observation that nanogram amounts of BBI, which is active against both trypsin and chymotrypsin, also suppressed neoplastic transformation caused by ionizing radiation through its ability to inhibit chymotrypsin (Yavelow et al., 1985). Moreover, chymotrypsin inhibition appeared to be responsible for suppression of the production of oxygen radicals by tumor promoter-induced human neutrophils. We noted that BBI was an order of magnitude more effective in suppressing superoxide formation by PM A-induced neutrophils than was the Kunitz soybean inhibitor, primarily a trypsin inhibitor (Yavelow et al., 1982). Further support for this hypothesis was obtained when Frenkel and Wiesner tested potato inhibitor 1(purified by C. Ryan), which specifi-

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WALTER TROLL E T AL.

cally inhibits chymotrypsin. Potato inhibitor 1 was by fhr the most effective inhibitor of H20z formation b y tumor promoter-activated neutrophils (Table I). Potato inhibitor 2, an inhibitor of both trypsin and chymotrypsin, was less inhibitory than potato inhibitor 1, but was more active than chicken ovoinhihitor, which also acts against both proteases (Frenkel et aE., 1986d).SBTI, which does not inhibit chymotrypsin, was practically inactive in suppressing H20Z formation (17% maximal inhibition at 80 pM).T h e potential for multiple biological actions of protease inhibitors that interfere with carcinogeiiesis requires consideration of some of these actions separately.

IV. Contribution of Oxygen Radicals to Carcinogenesis

A. DNA

DAMAGE

Reactive oxygen species generated by stimulated phagocytes might exert their activity by damaging or modifying cellular DNA (Emerit and Cerutti, 1981a). Birnboim et al. (Birnboim, 1982a,b, 1983; Birnboim and Biggar, 1982) have shown unambiguously that PMA induces extensive breaks in the DNA of PMNs through the action of oxygen radicals. Both superoxide dismutase (SOD) and catalase protected the DNA of PMNs from that damage. However, only catalase prevented the formation of breaks in the DNA of nioiise erythroleukernia cells that were coincubated with activated PMNs, thus indicating that it is HZ02 which is responsible for the DNA damage. This action of H202 is similar to the inactivation of excess sperm and the prevention ofpoly-

TABLE I PEHCEWT INHIMITION OF € 3 2 0 2 FOIIYATION BY

PMA-ACTIVATEDNEUTROPHILS Coiiceritration of protease iiihibitoi 1iihil)itor

5 pM

Potato inhibitor 1 Potato inhibitor 2 Chicken ovoinhibitor Soybean trypsin inhibitor

12 33

"

Not tested.

17

-

1 0 pLM 89 39 23

0.3

40 pLM

73

52 16

ANTICARCINOGENIC ACTION OF PROTEASE INHIBITORS

275

spermy in sea urchins (Coburn et al., 1981; Frenkel et aZ., 1986a). Protease inhibitors by blocking formation of HzOz cause polyspermy in fertilized sea urchin eggs (Coburn et al., 1981). One of the PMN-generated damaging agents causing DNA strand breaks and modification of bases may be the hydroxyl radical (.OH), which is formed by an interaction of 0; with HzOz in the presence of some transition metal ions (Frenkel et al., 1986b,c). If tumor promotion is in some aspects similar to the action of ionizing radiation, which is known to induce formation of *OH and of strand breaks in DNA, it also should cause the same type of modification of the DNA bases as does y-radiation (Frenkel et al., 1981, 1985; Teebor et al., 1982, 1984). Indeed, [3H]thymidine-labeled DNA incubated with PMA-stimulated PMNs in the presence of autologous plasma revealed the presence of 5-hydroxymethyl-2’-deoxyuridine(HMdU), a thymidine derivative known to be formed by y-radiation (Troll et al., 1984; Frenkel et aZ., 1986b,c). This derivative was formed in amounts equivalent to the amount of HMdU obtained with 30 krad of y-radiation (Teebor et aZ., 1984; Frenkel et al., 1985). In addition to HMdU, thymidine glycol, another known product of y-radiation, was also formed in coincubated DNA (Frenkel et al., 1986b,c). Another type of phagocytic cell, activated macrophages, caused formation of DNA strand breaks in cocultivated cells (Gensler and Bowden, 1983), as well as formation of saturated thymine derivatives (Lewis and Adams, 1985). These results support the hypothesis that the oxidative burst of phagocytes is capable of causing modification of DNA bases similar to those formed through the action of y-radiation. The products of the oxidative burst of stimulated PMNs were found to be not only mutagenic (Weitzman and Stossel, 1981; Barak et aZ., 1983), but also carcinogenic (Weitzman et al., 1985). Therefore, it appears that at least some of the properties of tumor promoters, expressed through the PMN-mediated generation of oxygen radicals, might be similar to those of initiating carcinogens in that they cause modification of DNA constituents. Since protease inhibitors prevent the formation of oxygen radicals, we would expect that formation of HMdU and thymidine glycol might also be suppressed by these agents. Active oxygen species participate in a variety of normal cell functions. These include glucose transport (Taylor and Halperin, 1979), activation of guanylate cyclase (White et d.,1976), and phagocytosis (Troll and Wiesner, 1985). In fact, a role for 0; and HzOz in normal cell division has been proposed (Oberley et aZ., 1981). Multiple defense systems directed against the excessive reactivity of these oxygen

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WALTER TROLL ET AL.

species exist in the cells. They include two recently found DNA glycosylases, repair enzymes which recognize and remove thymine glycol (Rreimer and Lindahl, 1985; Higgins et al., 1986), and 5-hydroxymethyluracil (Hollstein et d., 1984; Boorstein et aZ,, 1986). They also include SOD and catalase, which have been shown to block tumor promotion b y PMA and bleoniycin in hamster embryo and C3H 10Tl/2 cells (Borek and Troll, 1983; Kennedy et nl., 1984b). One of the consequences of treating mouse skin with PMA seems to be lowering of the levels of the two latter protective enzymes (Solanki et al., 1981).

B.

INDU(:’IlON OF

POLY(ADP-KIBOSE) FORMATION

Another consequence of tumor promotion by PMA, again perhaps through the generation of free oxygen radicals, is the induction of poly-ADP-ribosylation (Kobayashi et al., 1984; Singh et d.,1985). Recent evidence indicates that a close relationship exists between the formation of poly(ADP-ribose) and nuclear events involved in DNA damage and repair in mammalian cells (Suginiura and Miwa, 1983). Poly(ADP-ribose) synthetase (transferase, polymerase), the enzyme responsible for the formation of poly(ADP-ribose) from nicotinamide adenine dinucleotide (NAD), is activated in response to treatment of cells with D N A damaging agents. Treatment of normal cells with alkylating agents or with ionizing radiation causes a decrease in cellular NAD content with a concomitant increase in poly(ADP-ribose) synthetase activity (Shall, 1982). Cells obtained from patients with Fanconi’s anemia syndrome are extremely sensitive to the damaging effects of ionizing radiation (Remsen and Cerutti, 1976). These cells were shown to contain only very low levels of NAD (Berger et el., 1982). Protease inhibitors may play a role in controlling the formation of poly(ADP-ribose). The polymer itself, poly(ADP-ribose), is a chymotrypsin inhibitor; it was isolated from rat peritoneal macrophages and identified as poly(ADP-ribose) of 2,000-3,500 molecular weight containing four to seven ADP-ribosyl units (Inagaki et ul., 1980). More recently, it was shown that benzamide and 3-aminobenzamide, specific inhibitors of poly(ADP-ribose) synthetase, also reduced malignant transformation in C3H 10Tl/2 cells (Borek et ul., 1984b). Therefore, it was of interest to determine whether inhibitors of poly(ADP-ribose) synthetase such as benzainide, 3-aminobenzamide, arid nicotinamide were also inhibitors of the proteases trypsin and chymotrypsin. We have tested the effects that these inhibitors have on the hydrolysis of jH-labeled casein b y the two enzymes. As can be

277

ANTICARCINOGENIC ACTION OF PROTEASE INHIBITORS

TABLE I1 EFFECTOF VARIOUS INHIBITORS ON HYDROLYSIS OF [3H]CASEIN BY CHYMOTRYPSIN AND TRYPSIN

Inhibitor

Chymotrypsin"

10 20 40 60 10 20 40 60 10 20 40 60

22 53 52 57 64 76 88 34 50 69 72

Nicotinamide

Benzamide

3-Aminobenzamide

a

Inhibition (%)

Concentration of inhibitor (mM)

7

Trypsin" 6 12 14 40 24 37 44 64 6 27 31 40

Twenty nanograms chymotrypsin or trypsin per assay.

seen in Table 11, all three agents blocked the activity of both chymotrypsin and trypsin. However, in every case, chymotrypsin was more sensitive than trypsin to the action of these inhibitors (Wiesner and Troll, 1985). All three of these inhibitors block the production of 0; by human PMNs stimulated by PMA (Table 111) (Troll et al., 1986). If the induction of poly(ADP-ribose) is directly caused by the action of oxygen radicals, as has been asserted (Cerutti, 1985), it can be speculated that agents capable of blocking the formation of superoxide by phagocytic cells also may suppress the process of poly-ADPribosylation. Thus, it is possible that it is a protease inhibitor which provides the biological signal for controlling the formation of poly(ADP-ribose). This hypothesis was supported by a recent observaTABLE I11 INHIBITION OF SUPEROXIDE ANIONFORMATION BY HUMAN NEUTROPHILS Inhibition (%) Concentration of inhibitor

Nicotinamide

Benzamide

3-Aminobenzamide

2 mM 5 mM 10 mM

18 23 35

23 34 84

15 48 73

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WALTER TROLL ET AL.

tion that the protease inhibitors leupeptin and antipain inhibited poly-ADP-ribosylation in C3H 10TY2 cells (Cleaver et d.,1986; N. Singh and P. Cerutti, personal communication). Inhibitors of polyADP-ribosylation such as benzamide and 3-aminobenzamide block the transformation of cells in culture caused b y many alkylating agents, UV light, and PMA (Borek et id.,1984a). These inhibitors and other drugs that modulate the action of poly(ADP-ribose) synthetase also blocked the transformation of carcinogen-treated human fibroblasts (Milo et id.,1985)and of C3H 10TVz cells (Borek et d., 1984b). We have recently observed that even nanogram amounts of pure BBI suppressed the ionizing radiation-induced transformation of C3H 1OTlh cells (Yavelow et d.,1985). T h e minute amount of protease inhibitor needed suggested that its action is mediated by a receptor. Only that part of the Bowman-Birk inhibitor which inhibits chymotrypsin was required for blocking transformation. T h e known poly(ADP-ribose) inhibitors benzamide, 3-aniinobenzamide, and nicotinamide are also better inhibitors of' chyniotrypsin than of trypsin. Therefore, it is possible that the receptor for induction of poly-ADPribosylation is a chymotrypsin-type receptor. The full biological function of poly(ADP-ribose) is not a s yet known. It appears to play a role in DNA repair in mammalian cells (Sugimura and Miwa, 1983). It is possible that cancer cells are the unexpected beneficiary of this particular repair system which may have developed to assure the survival of cells at any cost. Thus, protease inhibitors by inhibiting the process of poly-ADP-ribosylation might counteract sonie of the promotional events of carcinogenesis. V. Effect of Protease Inhibitors on Selective DNA and RNA Amplification

We have described numerous examples of suppression of carcinogenesis b y protease inhibitors and many biological reactions possibly related to this effect. Recently, a direct effect of protease inhibitors on initiator-induced amplification of DNA was observed (Heilbronn et d.,1985).Inhibitors of D N A polymerase cy blocked initiator-induced selective DNA amplification (SDA) in the SV40-transformed Chinese hamster cell line (C0631) suggesting a specific role for cellular DNA polymerase a in carcinogen-induced SDA. I n addition, protease inhibitors including &-aminocaproicacid (1.35mg/ml), q-antitrypsin (17 pglml), leupeptin (100 pg/ml), antipain 40 pglml), and soybean protease inhibitor (40 pg/ml) successfully blocked SV40 DNA amplification induced by the carcinogens DMBA and N-methyl-N'-nitro-N-nitrosoguanidine or by HSV-1 infection. Surprisingly, DNA amplification

ANTICARCINOGENIC ACTION OF PROTEASE INHIBITORS

2 79

resulting from cycloheximide treatment occurred even when protein synthesis was completely blocked. It was suggested that modification of DNA polymerase a by proteolytic cleavage could have been responsible for carcinogen-induced S DA. Protease inhibitors may reduce the extent of SDA induction by preventing the modification of DNA polymerase (Y (Heilbronn et al., 1985).Another example of protease inhibitors’ interference with a selective DNA function was noted by Chang et al. (1985); it was shown that myc expression was inhibited by antipain whereas cellular growth or total RNA synthesis was not affected. This group has also shown that myc expression was blocked by BBI and chymostatin (A. R. Kennedy, personal communication). The effect of protease inhibitors on DNA and RNA replication, as shown b y the above examples, may represent the basic principle b y which these inhibitors suppress and modulate the development of carcinogenesis. This is an exciting possibility which requires further confirmation in animal and tissue culture systems where protease inhibitors were shown to suppress carcinogenesis. The possibility that chemopreventive agents act through suppressing oncogene expression opens new opportunities for identifying and measuring the anticarcinogenic action of nutrients in our diet. VI. Summary

Protease inhibitors are synthesized in biological systems and play a critical role in controlling a number of diverse physiological functions. They participate in blood clotting and lysis of clots, in growth processes by modulation of proteolytic digestion of proteins and thus availability of amino acids, and in the induction of selective DNA amplification. When incorporated into the diet, protease inhibitors appear to suppress many types of cancer. In vitro, they suppress neoplastic transformation caused by chemical carcinogens, ionizing radiation, and oncogenes. These observations offer the hope that judiciously applied protease inhibitors in small concentrations may prevent a wide range of human cancers. This hope is further supported by epidemiological studies which show that populations consuming relatively large amounts of protease inhibitors have a lower occurrence of cancer. The tasks remaining are to determine the kind and the level of protease inhibitors that are most effective in preventing cancer without also having toxic side effects and to incorporate them into our diet. Perhaps the most encouraging investigations are those using small

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nontoxic protease inhibitors available in pure form (c-aniinocaproic acid, a trypsin plasminogen activator inhibitor, and nicotinamide, a chymotrypsin inhibitor and known vitamin). Both agents have been shown to be preventive agents of cancer in animals and in 0itt-o models. Further studies with natural protease inhibitors may yield even more effective agents which when incorporated into our diet will prevent the development of ntany types of cancer.

ACKNOWLEDGMENTS This investigation was supported in part by Pithlic Health Service grants (CA 16080 and CA 37858) and Center Grant No. CA 13343, awarded b y the Natiorial Canccr Institute, Department of Health and Hnrnan Services, Center Grant No. ES 00260, front the National Institute of Environmental Health Sciences, and Special Institiitional Grant No. oooO9, from the American Cancer Society. We gratefully acknowledge the help of Susan Benninghoff in editing and preparing the manuscript.

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ON THE EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE Ross L. Prentice and David 6.Thomas Division of Public Health Sciencss. Fred Hutchinson Cancer Research Center, Seattle, Washington, 98104

I . Introduction

The purpose of this article is to review and assess the epidemiologic literature on the association between the use of oral contraceptives (OCs) and subsequent disease risk. This literature is replete with reports of both adverse and beneficial effects in relation to a range of human diseases including a number of common cancers and important cardiovascular diseases. This review will proceed by sequentially examining important disease categories with emphasis on the consistency of results and, when possible, on the apparent form of any increased or decreased risk as a function of such “exposure” features as hormone content of OC used, duration of use, and time since first and last use of OCs. Topics that seem particularly important for further study will be indicated throughout, and an overall risk-benefit summary, with an emphasis on mortality rates in the United States, will be attempted. Steroid contraceptives contain an estrogen or a progestogen, or a combination thereof. By far the most commonly used steroid contraceptive is the combined OC in which each pill contains both an estrogen and a progestogen. Such pills are to be taken daily for 3 weeks and then use is ceased for 1 week, withdrawal bleeding occurs, and the cycle is resumed. Such preparations suppress ovulation and, in the unlikely event that ovulation occurs, evidently reduce conception rates by affecting ovum transport through the fallopian tubes and by altering cervical mucus so that reduced spermoidal penetration occurs. These products were first marketed in the United States in the early 1960s. Their use rapidly spread throughout the world, and by 1980 approximately 80 million women had used combined oral contraceptives. The original Enovid pill, containing 10 mg of norethynodrel (a progestogen) and 150 pg of mestranol (an estrogen), is still 285 ADVANCES IN CANCER RESEARCH, VOL 49

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marketed, but most current usage involves 1mg or less ofprogestogen and 50 mg or less of an estrogen. The specific estrogen and progestogen used varies among a considerable number of preparations. In the United States norethindrone is a commonly used progestogen and ethinyl estradiol is the most commonly used estrogen. (see Oiy et al., 1983, for a list of preparations marketed in the United States as of

1983). Sequential oral contraceptives were marketed widely in the United States up to a few years ago and are still in use in some countries. These preparations involve exposure to estrogen alone for approximately 15 days of a woman’s menstrual cycle, approximately 5 days of an estrogen plus a progestogen, followed by cessation for a week with resultant withdrawal bleeding. Because of the substantial period of “unopposed” estrogen with such sequential preparations one may gain valuable insights into the hormonal aspects of disease occurrence by comparing the epidemiologic effects of sequential and combined OCs. Such comparison might also lead to hypotheses concerning the effects of other steroid contraceptives, such as the minipill, for which few epidetniologic data currently exist. The minipill has been sold in the United States since 1973. Such pills contain only a progestogen, and at a considerably lower dosage than is found in the combined pill. This pill does not suppress ovulation but appears to act by creating a thickened cervical mucus and by inhibiting ovum transport and implantation. This pill is not currently widely used. Some other strongly progestational contraceptives, particularly long-acting injectable preparations, such as norethindrone enanthate and, especially depo-medroxyprogesterone acetate (DMPA), have been used extensively in a large number of countries (e.g., Thailand, New Zealand, Sri Lanka, and Jamaica). DMPA, for example, is usually administered every 3 months in a dose of 150 mg. These products are not currently licensed for use as contraceptives in the United States. Study of their relationship to disease occurrence provides direct insight into the role of progestogens in human disease. A review of the epidemiologic data relating to OC use seems appropriate at this time. Public sensitivity to possible risks associated with drug exposure leads to wide dissemination of each report concerning hazards of oral contraceptive use. Such reports do not always stand up to subsequent confirmation in more rigorous investigations, however, and reports of benefit may not be so widely disseminated or appreciated. Furthermore, rather few attempts have been made to draw together the findings in respect to such major disease categories as cancer and cardiovascular disease. Of course, the ability to carry out such

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assimilation from published data is somewhat limited, and observational studies are, by their very nature, subject ot biases that may be of the same order of magnitude as are the associations being studied. Observational studies of OC use need particularly to recognize the changing dosage and composition over time in available preparations as was mentioned above, and to recognize that contraceptive choices are undoubtedly influenced by perceived risks. The next section amplifies these latter points in the context of a more general discussion of study methods. II. Study Methods

The studies reviewed in this report are all observational: Decisions concerning whether or not to use OCs and concerning the “pattern” of such use are made subjectively by the women on whom data are collected. In order to identify any disease risk associated with OC usage one then requires auxiliary data on factors that may affect such decisions and that are also relevant to disease risk. The identification of such “confounding” factors involves considerable knowledge of disease causation and of contraceptive decision making. It is usual to collect data on only a handful of the most likely, or the most readily available, potential confounding factors. Hence any reported association from an observational study will be subject to some uncertainty concerning causality. Associations that are strong, that exhibit “regular” dose-response relationships, and that can be replicated in a range of study populations come, in time, to be regarded as causal. There are a number of other potential sources of bias in observational studies. For example, in cohort studies the accuracy with which confounding factor data are obtained and, perhaps more importantly, the accuracy and completeness with which disease events are ascertained may depend on a subject’s OC use history. Furthermore, such histories themselves may be ascertained with noteworthy inaccuracy. Additional important issues in case-control study design include the ability to select a random sample of the cases and of the controls arising from the population under study (selection bias) and the ability to obtain equally accurate information on oral contraceptive usage and on auxiliary variables from both cases and controls (recall bias). See, for example, Horwitz and Feinstein (1979) for an inventory of such issues. Data analysis procedures also play an essential role in the proper evaluation and reporting of observational studies. In fact, the need to take appropriate account of detailed exposure and auxiliary variable

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histories is more important in observational studies than, say, in randomized trials. That such histories are often reduced to simple dichotomous or polychotomous variables prior to data analysis introduces another potential source of bias. Misclassification, or measurement error more generally, may also have important effects on the strength of the relationship between some measure of OC use and subsequent disease. Typically no formal adjustment for measurement errors is made in the reporting of epidemiologic studies, thereby giving rise to some distortion (usually dilution) of the strength of association and to possible heterogeneity among the results of different studies. The relative risk, or more generally the relative risk process, provides a convenient basis for the summary and comparision of reported associations between oral contraceptive use and the occurrence of specific diseases. Such relative risks are often more stable across study subject characteristics and across study populations than are corresponding absolute risks, for example. Furthermore, relative risks or the closely associated odds ratios, but not absolute risks, can be directly estimated from case-control studies. Of course, recognition of the magnitude of the “baseline” rates for the diseases under study is essential for an understanding o f t h e importance of an observed association and for the assembly of results across disease categories. Sections 11, A-C introduce notation that allows many of the above points to be made more explicitly. This material is somewhat technical and may be skipped without affecting the readibility of the remainder of the paper.

A. RELATIVE RISK Consider a conceptually infinite population of women about which inferences are to be drawn concerning oral contraceptive use and the occurrence of a specific disease. For example, such a population may consist of all female residents of a certain geographic area who are within a specified age range. Let x ( t ) summarize a subject’s oral contraceptive use history up to age t. For example, one may define x ( t ) = 0 if the subject has never used OCs prior to age t , x ( t ) = 1 if the subject is using OCs at age t and x ( t ) = 2 if the subject has used OCs prior to, but not at, age t. More generally x ( t ) may be defined as a fixed length vector that includes aspects of dosage, duration, and consistency of OC use. One may also define the subjects stratum assignment, s = s ( t ) , at age t in terms of potential confounding factors pertinent to age t or earlier. For example, one may define s(t) = 1 if the woman had never been a cigarette smoker at age t or earlier, s ( t ) = 2 if the woman is a

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former smoker, s ( t ) = 3 if a light smoker and s ( t ) = 4 if a heavy smoker, at age t . More generally the stratification may depend on several factors; for example, parity, social class, and cigarette smoking habits. The relative risk associated with a general OC vector x ( t ) compared to that for a never user [ x ( t )= 01 in stratum s is then defined by RRs{x(t)} = h{t;x(t),s(t)}/h{t;O,s(t)) where X{t;x(t),s(t)} is the (instantaneous) disease rate for a subject at age t with OC history characterized by x ( t ) and with stratum s ( t ) , at age t . The relative risk will be independent of factors included in the stratification definition if the disease rate is independent of such factors or if such factors are independent of x(t), conditional on the other stratification variables. Such factors are termed “nonconfounding” in common epidemiologic parlance. Note that factors that respond to OC usage should, in general, not be included in the stratification since the relative risk of interest would then typically be underestimated. For example, some studies mentioned below stratify on diagnosed hypertension, even though OCs stimulate an elevation in average blood pressure levels. The proper use of hypertension information in such analyses is a rather thorny issue, however, because hypertension is an important risk factor for certain disease categories reviewed below, and OC use is likely to be less common among women with elevated blood pressure.

B. COHORT STUDIES Suppose now that a well-defined cohort is selected from the population under study. For example, such a cohort may include all female enrollees in a health maintenance organization who are within a specified age range at a certain point in chronological time. In order to make valid inferences on population disease rates the cohort needs to be able to be viewed as an independent random sample from the population of interest, given the baseline information collected on cohort members. For a given cohort member some of the data on OC use and on stratification factors may pertain to ages prior to the woman’s selection into the cohort and hence will need to be obtained retrospectively by interviews or other means. An important advantage of the cohort study, however, is that such data pertaining to ages after cohort identification can be obtained prospectively during the course of cohort follow-up and, most importantly, that all relevant covariate data are typically obtained prior to disease diagnosis. Issues in the design and conduct of cohort studies include the ability to obtain

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detailed and accurate OC usage histories and potential confounding factor histories, and the ability to ascertain all relevant disease events of interest among cohort members under active follow-up. Clearly estimates of the relative risk defined above can be expected to be biased unless the disease detection rates are independent of the preceding oral contraceptive histories. Now consider, briefly, the estimation of relative risks in cohort studies. Most of the reports reviewed below group the data into age intervals and assume that disease rates are approximately constant within an age interval t , given the corresponding OC variable x ( t ) and the stratification variable s ( t ) .The relative risk corresponding to any specific OC use category [value of x ( t ) ] is aiso usually taken to be independent o f t (age) and s ( t ) , whence a simple relative risk estimate is obtained as the ratio of the indirectly standardized disease rate for the OC category in question to that for the “never-use” OC category. Such an indirectly standardized rate is simply a linear combination of the crude disease rates, defined as the ratio of the number of events to the number of person-years of observation, across strata (including age intervals). The weighting factors are defined by the number of subjects under active follow-up in a given stratum in the entire cohort. The logarithm of such a relative risk estimate typically adheres approximately to a normal distribution with only a moderate number of disease events. A simple variance formula for this logarithm then leads to a convenient approximate confidence interval for the relative risk. T h e precision with which a specific relative risk can b e estimated depends primarily on the number of events and the total person-years at risk in the OC categories being compared. Such information is therefore given in the subsequent cohort study tables. Relative risks may be displayed on a stratum-specific basis in order to test, and relax as appropriate, the assumption of common relative risks across strata. In the past 15 years or so a considerable literature has developed concerning the use of relative risk regression methods (e.g., Cox, 1972; Prentice and Farewell, 1986). Such methods provide potential for a more thorough use of the details of OC histories and corresponding confounding factor histories without age grouping than is possible using the standardized rates described above. It may be helpful to apply such methods to the cohort studies described below.

c. CASE-CONTROL STUDIES Consider again the conceptually infinite population about which inferences are to be drawn. Rather than select a cohort for follow-up a case-control study proceeds by relying on a system, such as a popula-

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tion-based registry, for identifying all or a random sample of incident cases of disease in the population. OC use and stratification factor histories are then obtained retrospectively, both for the selected cases and typically for a random sample of disease-free individuals. For example, a case-control study may attempt to enroll all cases of a certain cancer site newly diagnosed within a certain period of time that enter a population-based cancer registry. Though there are many variations on control choice, and on the specifics of case-control design more generally, suppose here that, for each case, one or more controls are randomly selected from the subset of the population that is without disease at the age of case diagnosis in the same stratum as the case. That the relative risk process given above is identifiable from such data arises from the fact that

where T = t denotes a case occurrence at age t, while T > t denotes the absence of disease at age t or earlier, and p r , denotes probability in stratum s. The “instantaneous” odds ratio on the right-side of this expression is directly estimable on the basis of age-matched casecontrol data (e.g., Prentice and Breslow, 1978). One of the most serious concerns in a case-control study relates to the ability to collect retrospectively detailed and accurate histories on OC use and stratification factors. Specifically, there is often concern that diagnosis of a serious disease will sensitize the cases to exposures or risk factors that are perceived to be pertinent to their disease thereby creating a recall bias relative to the controls. The potential for bias may be compounded if data collection personnel are aware of the subject’s disease state. In terms of the above notation such heterogeneity would give rise to biased estimation of

pr,{x(t)lT = t}/pr,{x(t)lT> t} and hence of the relative risk. Other important issues concern the ability to sample randomly from cases arising in the population and, particularly, to sample randomly from controls in the same population from which the cases arise. The issues of confounding factor control, of random errors in the OC or stratification factor histories, and of possible dependence of the completeness of disease diagnosis procedures on OC histories are as important for case-control as for cohort studies. In many of the casecontrol studies reviewed below controls are selected from among pa-

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tients with diseases other than that under study. This method can also yield valid relative risk estimates provided control diseases are unrelated to OC use, that cases and controls arise from the same population, and provided careful account is taken of risk factors for both case and control diseases. Consider, briefly, the estimation of relative risks from case-control studies: If relative risks are assumed independent of age and other matching factors some simple matched relative risk estimates can be calculated. For example, if there is exactly one matched control per case the relative risk for a specific OC characteristic x ( t ) = x is merely the ratio of the number of pairs in which the case had OC value x while the control had zero divided by the number of pairs in which the control had OC value x and the case zero. The logarithm of this ratio has asymptotic variance estimator the sum of the reciprocals of these two numbers. More commonly cases and controls with similar ages at ascertainment will be grouped together in which circumstance an estimate of the odds ratio in a particular stratum (including age interval) is readily estimated by the product of the number of cases with OC value x multiplied b y the number of controls with value zero, divided by the product of the number of controls with OC value x and the number of cases with value zero. The logarithm of this odds ratio estimate has asymptotic variance estimator given by the sum of the reciprocals of the four numbers just mentioned. A summary odds ratio estimator can be obtained by combining such stratum-specific odds ratio estimators (e.g., Mantel and Haenszel, 1959). The precision with which such a summary odds ratio can be estimated depends primarily on the total number of cases and the total number of controls that are never-users of OCs or that have the OC value (x) under study. Hence these numbers are given in the subsequent case-control study tables. Odds ratios will closely approximate the corresponding relative risk provided the disease is rare in both exposure groups. A considerable regression literature has also arisen for the analysis of case-control studies, mostly centered on the use of logistic disease probability models (reviewed in Prentice and Farewell, 1986). A few of the studies reviewed below made use of such logistic regression mode 1s.

D. LITERATURE REVIEWPROCEDURES The above discussion and notation suggests an ideal in observational study design, conduct, and analysis. One would like accurate and detailed histories of oral contraceptive use with a sufficient sam-

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ple size and structure so that such aspects as preparation, dosage, and duration of usage can be assessed in relation to disease occurrence, and so that the strength of any association can be studied as a function of study subject characteristics. One would like thorough and accurate data collection in respect to all important confounding factors along with a sample size and structure that would permit a detailed use of such information in data analysis. One would like a procedure for disease diagnosis that is complete and accurate and, most importantly, for which the completeness and accuracy does not depend on oral contraceptive usage. Finally, one would like key hypotheses and data analysis procedures to be specified in advance in order to avoid the pitfalls and anomalous results of multiple significance testing. Relative to such ideals one can attempt to assess qualitatively for each study whether certain criteria appear to have been met, as has been recently done in a very detailed manner for oral contraceptives and cardiovascular diseases (Realini and Goldzieher, 1985), and one can attempt to assess, perhaps more quantitatively, the degree of consistency among studies of a specific association. Of particular interest will be a comparison of the consistency of the results from cohort and from case-control studies, since rather different sources of bias are potentially important. Hence summary relative risk estimates across studies are presented separately for cohort and case-control studies in the subsequent literature review. For a quantitative assessment of consistency among studies the common information that can usually be readily extracted from published reports includes an estimate (dR)of relative risk for certain definitions of oral contraceptive usage (e.g., current, former, and never) for each disease category, along with a confidence interval for such relative risk. When common definitions are used in two or more studies one can test equality of the underlying relative risks among studies and, under a hypothesis of equality, can produce a summary relative risk estimate and confidence interval. More explicitly, let = log RRj be the logarithm of the relative risk estimate for the j t h study out of k studies of a particular association. Except in modest samples the standard error of is well approximated by

Bj

bj

A

aj = log{RRj/Lower 95% confidence limit RRj}/1.96 or equivalently by A

uj = log{Upper 95% confidence limit RRj/BRj}/1.96

(The average of these two was used unless a standard error estimate of Bjwas reported.) An asymptotic xf-1test of the hypotheses of equality of the RRj, j = 1, ., k is then given by

..

294

ROSS L. PRENTICE AND DAVID R. THOMAS

where

A

A combined study relative risk estimate is given by RR = ea with a corresponding asymptotic 95% confidence interval of {exp@ - 1.96a), exp@ + 1 . 9 6 ~ )where ) k j=l

Relative to other methods of combining studies the above procedure has the advantage of retaining the standardization, stratification, or regression modelling that was used in the calculation of relative risk estimates in the individual study reports. In some circumstances the subsequent displays combine the disease categories used in previous reports. For cohort studies the standardized incidence rates were simply added across the relevant disease categories, possibly resulting in a very small overestimation for nonfatal event rates (since a specific subject may experience events in more than one category). Relative risk estimates following such combinations are readily obtained using the methods described above, but corresponding variance estimates may not be available from the reports under review. The following procedures were used whenever a confidence interval for the relative risk or, equivalently, a variance estimate for the logarithm of the relative risk estimate was unavailable: For a cohort study the variance of the logarithm of the relative risk estimate was approximated by the sum of the reciprocals of the number of disease events in the two OC use categories being compared. For a casecontrol study the variance of the logarithm of the odds ratio estimate was approximated by the sum of the reciprocals of the case and the control counts in the two OC use categories being compared. In a few situations the reported odds ratio was based on a matched pair analysis. If a corresponding confidence interval was not given the variance of the logarithm of the odds ratio estimate was approximated by the

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295

sum of the reciprocals of the numbers of discordant pairs mentioned above. In each circumstance these approximations would be wholly justifiable if the data were confined to a single (narrow) age interval and stratum. The additional variation introduced into these estimates by standardization or other means of confounding factor control seems likely to be minor in view of generally weak associations between OC use and control factors, except possibly when the number of disease events is small. The asymptotic confidence interval procedures used in the original reports and here are likely to be somewhat anticonservative (i.e., to give slightly too narrow intervals) with greater anticonservatism in the presence of small numbers of disease events in either of the OC categories being compared. Our literature review is confined to cohort and case-control studies. For brevity, less analytic studies describing, for example, time trends in disease incidence or mortality are not considered. Ill. Oral Contraceptives and Mortality: An Overview

In 1968 three substantial cohort studies were initiated to study the relationship between oral contraceptive use and subsequent disease occurrence. The Royal College of General Practitioners’ (RCGP) Oral Contraception Study enrolled 46,000 women in 1968 and 1969 (RCGP, 1974). Enrollees were aged 15 or older with about 8% aged 40 or older. Subjects were equally divided between oral contraceptive users and never-users. Twice per year the 1,400 participating general practitioners in the United Kingdom report ori the morbidity and mortality of their study subjects. The Oxford Family Planning Association Contraceptive Study recruited 17,032 women during 1968-1974 at 17 British family planning clinics (Vessey et al., 1976). Enrollees were 25-39 years old. Fifty-six percent of the cohort were oral contraceptive users while the remainder were diaphragm users (25%) or intrauterine device users (19%). The Walnut Creek Contraceptive Drug Study (Ramcharan et al., 1981) enrolled 16,638 women aged 18-54, who were members of the Kaiser Foundation Health Plan in Northern California, between 1968 and 1972. About 40% were 40 years of age or older. At recruitment 28% of the women were current oral contraceptive users, 33% were former users, and 39% were never-users. Active follow-up of this cohort terminated at the end of 1977. Data from these three cohorts will be referred to frequently in subsequent sections. Here as general introduction to the potential health consequences of OC use we shall examine the overall and causespecific mortality reported for these cohorts. There were 249 deaths in

296

ROSS L. PRENTICE AND DAVID B. THOMAS

the RCGP study group up to the end of 1979 (RCGP, 1981a). This period included 98,997 person-years of current OC use, 84,811 of former use, and 138,630 person-years of never-use. Cause-specific mortality rates were Standardized indirectly according to age and parity at death and according to cigarette use and social class at study entry. There were 81 deaths based on 79,678 person-years of followup in the group taking OCs at entry and 62,532 person-years of followup in the group using a diaphragm or intrauterine device in the Oxford Family Planning Association Study (Vessey et d., 1981a). Mortality rates were standardized indirectly for age, parity, social class, and smoking habits. The Walnut Creek Study Group reported mortality data up to the end of 1977, including the analysis of 138 deaths and 127,490 person-years of observation (Ramcharan et al., 1981, Ch. 9). Mortality rates were indirectly standardized for age, parity, cigarette smoking, education, and (menopausal) estrogen use. Section (a) of Table I shows total mortality relative risks from the three studies for ever-users of oral contraceptives versus never-users. Ever-users in the RCGP study experienced a 40% greater (standardized) mortality rate than did never-users, an increase that is significant at the 0.05 level. The Oxford study first reported (Vessey et aZ., 1977b) a 20% increase in total mortality based on 43 deaths, but their analysis based on the 81 deaths indicated in Table I shows a nonsignificant 10% decrease. The Walnut Creek study shows an overall nonsignificant 10% increase in total mortality among ever-users of oral contraceptives. The x2 test of equality of the three relative risks (see Section I1,D) gives a significance level of 0.18. The combined study relative risk estimate takes value 1.2 with an approximate 95% confidence interval from 1.0 to 1.4. Note that the standardized mortality rates in Table I are not directly comparable across studies since different standardization coefficients were used in each study. Even though there is not significant evidence of relative risk heterogeneity it is of interest to speculate on the source of differences among the observed relative risks. The three studies began at about the same time and had similar follow-up periods in their 1981 reports. Hence changes in oral contraceptive composition and dosage over time are likely to affect the three relative risks similarly. The Walnut Creek study involved a large number of former OC users at cohort identification. Such usage will generally correspond to relatively high doses, but corresponding exposure durations tended to be relatively short, particularly among older women. Of course any effects on mortality rates may be quite different among current as compared to former OC users.

TABLE I ORALCONTRACEPTIVES AND MORTALITYIN THREE COHORT STUDIES Cases per 1000 person-years (number of cases) Source

Ever-users

Never-users

RR estimate (95% c14)

(a) Total mortality RCGP (1981a) Vessey et al. (1981a) Ramcharan et al. (1981)

0.87 (156) 0.53 (40) 1.78 (66)

0.64 (93) 0.62 (41) 1.53 (72)

1.4 (1.1,1.8) 0.9 [0.6,1.31 1.1 [0.8,1.51b

(b) All mortality from diseases of the circulatory system (ICD 390-458) 0.07 (10) 4.2 (2.3,7.7) RCGP (1981a) 0.30 (55) Vessey et al. (1981a) 0.12 (10) 0.05 (3) 2.5 [0.7,9.1] Ramcharan et al. (1981) 0.25 (7) 0.33 (15) 0.8 [0.3,1.91

Heterogeneity (p value)

Combined RR estimate (95% CI)

(p = 0.18)

1.2 [1.0,1.4]

( p = 0.01)

2.5 [ 1.6,4.0]

(c) All cancer mortality (ICD 140-209) RCGP (1981a) 0.30 (53) 0.25 (18) Vessey et al. (1981a)" Ramcharan et al. (1981) 0.96 (29)

0.32 (46) 0.39 (28) 0.69 (32)

1.0 (0.5,l.g) 0.6 [0.4,1.21 1.4 [0.8,2.3]

( p = 0.10)

1.0 [0.8,1.3]

(d) All other mortality RCGP (1981a) Vessey et al. (1981a) Ramcharan et al. (1981)

0.24 (37) 0.17 (10) 0.44 (25)

1.1 (0.7,1.7) 0.9 [0.4,2.01 1.2 [0.7,2.1]

( p = 0.85)

1.1 [0.8,1.5]

0.27 (48) 0.15 (12) 0.54 (30)

( ) Implies confidence interval given in reference cited; [ 3 implies approximate confidence interval calculated by us using approximate methods described in Section 11. The age groups of Table 9.3 of Ramcharan et al. (1981) were assigned weights 0.23,0.31, and 0.46, respectively, in calculating relative risks. ICD 140-239 (all neoplasms). (1

298

ROSS L. PRENTICE AND DAVID €3. THOMAS

Reporting procedures may also be important in explaining differences among observed relative risks. In the RCGP study a control woman who begins taking oral contraceptives contributes to the everuser group from the month at which such use began. The reports cited for the other two studies, however, describe ever- and never-use based on OC use history at the time of entry into the study. Hence some of the deaths in the never-user group may have occurred after the woman began taking oral contraceptives. The resulting “misclassification” between ever- and never-users would be expected to give rise to reduced (i.e., closer to unity) relative risks compared to the RCGP study, as is observed in Table 1. It is difficult to assess the completeness of the mortality data in these studies. Age-specific mortality rates are lower than corresponding national rates because subjects tend to be healthier, to be under more frequent medical surveillance, and to be of higher social class than the general population, and because, in the RCGP and Walnut Creek studies, deaths due to diseases prevalent at baseline were excluded. For example, in the Oxford study the standardized all-causes mortality rate was only 53% of the corresponding age-standardized mortality rate for England and Wales. This ratio was somewhat higher (73%) for neoplasms than for other major cause of death categories. Note, however, that the use of the National Health Service central registries in the two British studies should avoid any serious underascertainment of deaths, Whether or not a similar claim can be made for the Walnut Creek study is unclear. The total number of deaths occurring in that study was only 48% of that expected based on United States age-specific mortality rates, with the observed cardiovascular disease mortality being only 27% of that expected. Underascertainment of deaths may have been serious in this cohort, particularly since subjects moving out of the State of California were evidently not followed systematically. A x2 test of equality of relative risks in the two British studies is nearly significant ( p = 0.06). A comparison of the two cohorts using the same standardization, reporting, and follow-up periods may help clarify this issue. Section (b)of Table I gives results from the three studies for OC use in relation to mortality from all diseases of the circulatory system. The RCGP study, based on a moderate number (65)of deaths, gives an estimated relative risk of 4.2 which is highly significant. The Oxford study is fairly consistent with such an association but is based on only 13 deaths. The Walnut Creek study, on the other hand, is not suggestive of any increased mortality risk from diseases of the circulatory

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

299

system even though, as will be described below, an increased incidence of certain circulatory diseases (e.g., subarachnoid hemorrhage, venous thromboembolism) among OC users in this cohort has been reported. The summary relative risk for this cause of death category is 2.5 with an approximate 95% confidence interval from 1.6 to 4.0.Such a summary measure is, however, of questionable interpretation since there is significant evidence ( p = 0.01) of heterogeneity among the three relative risks. It seems reasonable to look to ascertainment issues with the Walnut Creek study and to differences in relative risk between current and former OC users for at least part of the explanation of such heterogeneity. Circulatory disease incidence and mortality will be discussed in detail in Section IV. Malignant neoplasms constitute the other major disease grouping that have been thoroughly studied in relation to OC usage. Part (c) of Table I is not suggestive of any overall difference in mortality rate from malignant neoplasms between ever- and never-users of oral contraceptives. In fact, the summary relative risk estimate based on the three studies is 1.0 with an approximate 95% confidence interval from 0.8 to 1.3. Such a coarse examination of the association between OC use and cancer may, however, obscure many important facts. For example, OC use may affect the incidence of certain malignant neoplasms that are rarely fatal or that constitute a small fraction of total cancer mortality. The incidence of certain cancers may be elevated while that in others is reduced, among OC users. Also, more recent OC preparations with their reduced estrogen and progestogen content may have quite different implications for cancer incidence and mortality than did earlier preparations. These topics will be taken up in Section V. The final section of Table I shows that there is no evidence from these studies for a change in the mortality rate from “all other causes” among ever-users versus never-users of oral contraceptives, nor is there evidence of heterogeneity among the three studies with respect to this mortality category. In summary, Table I provides evidence of somewhat elevated mortality among ever-users of OCs. The elevation may be of the order of 20%, or more when OC usage misclassification and issues of mortality ascertainment are taken into account. Virtually all of the increase appears to pertain to diseases of the circulatory system. As mentioned previously, much of the OC exposure pertinent to Table I involved preparations with considerably greater doses of both estrogens and progestogens than has been usual in recent years. The mortality and morbidity associated with more recent preparations is of

300

ROSS L. PRENTICE AND DAVID B. TIIOMAS

particular current interest. Also, Table I compares ever- and neverusers of oral contraceptives. One would like to be able to assess the risks and benefits of a range of OC usage patterns. Such issues as the dependence of risk alteration on the duration of OC use and on time since cessation of OC use are of considerable practical interest in relation to prescription practices. Subsequent sections will assess and summarize the literature on such topics. This will be done for diseases of the circulatory system (Section IV), malignant neoplasms (Section V), and other diseases (Section VI), after which a summary and an indication of further research needs will be given. IV. Oral Contraceptives and Disease of the Circulatory System

Most of the circulatory system mortality described above can be categorized further as either cerebrovascular disease, ischemic heart disease, or peripheral vascular disease. For example the 65 circulatory system deaths in the RCGP (1981a) report included 34 deaths (52%) from cerebrovascular diseases, 17 deaths (26%) from ischemic heart disease, and 5 deaths (8%) from pulmonary embolism. Hence we concentrate on these disease categories in this section.

A. CEREBROVASCULAR DISEASE

I. RelatiGe Risks Associated with Current and Former OC Use Table I1 summarized cerebrovascular disease incidence and mortality by OC use category for the three cohort studies described previously. Part (a) is concerned with all cerebrovascular disease. The RCGP (1983) report includes a11 events up to the end of 1979. Incidence rates are standardized as described previously (Section 111)for the mortality analyses. The Oxford Family Planning Association report (Vessey et al., 1984) involved cerebrovascular events accumulated by the end of January, 1984. Incidence rates were standardized for age, history of hospital referral for hypertension, and cigarette smoking. The Walnut Creek report (Ramcharan et al., 1981) included events up to the end of 1977 with standardization as described previously (Section 111). With the inclusion of incident cases each of the three studies provides evidence of an increased risk of cerebrovascular disease among current users. In fact the three relative risks are quite consistent ( p = 0.68 for x2 test of homogeneity) giving rise to a combined relative risk estimate of 2.9, with 95% confidence interval from 2.0 to 4.1. The inclusion of hospital referral for hypertension in

TABLE I1 AND CEREBROVASCULAR DISEASE: COHORT STUDIES~ ORALCONTRACEPTIVES Cases per 1000 person-years (number of cases) Source

Current users

Former users

Neverusers

RR estimate (95% CI) Current users

Combined RR estimate (95% CI)

Former users

Current users

Former users

2.9 [2.0,4.11 ( p = 0.68)"

1.8 [1.3,2.61 (p = 0.11)

2.0 [1.1,3.6] ( p = 0.13)

2.1 [1.1,3.91 ( p = 0.97)

3.8 [2.4,6.11 ( p = 0.21)

1.9 [1.2,3.01 (p = 0.01)

~~

(a) All cerebrovascular disease (ICD 430-438) 0.62 (51) 0.50 (46) RCGP (1983) 0.40 (17) 0.19 (14) Vessey et al. (1984)c 0.96 (7) 0.39 (16) Ramcharan et al. (1981)d All fatal cerebrovascular disease (ICD 430-438) RCGP (1981a) 0.10 (8) 0.18 (19) (b) Subarachnoid hemorrhage (ICD 430) RCGP (1983) 0.14 (11) Vessey et al. (1984) 0.06 (3) Ramcharan et al. (1981) 0.43 (4) Fatal subarachnoid hemorrhage (ICD 430) RCGP (1981a) 0.07 (6) Cerebral hemorrhage (ICD 431) RCGP (1983) 0.03 (2)

0.20 (26) 0.18 (13) 0.28 (17)

3.1 (2.0,4.9)b 2.2 [1.1,4.6Ib 3.4 [1.4,8.3]

2.6 (1.6,4.1) 1.1[0.5,2.2] 1.4 [0.7,2.81

0.05 (7)

2.0 (0.6,6.2)

3.6 (1.5,8.5)

0.17 (16) 0.09 (7) 0.10 (5)

0.08 (11) 0.05 (3) 0.04 (2)

1.7 (0.8,3.8) 1.2 [0.2,5.9] 10.1 (1.8,55.1)

2.1 (0.9,4.6) 1.8 [0.5,7.0] 2.3 (0.4,ll.g)

0.10 (11)

0.02 (3)

3.2 (0.6J6.3)

4.5 (1.2,16.5)

0.09 (8)

0.03 (4)

1.1(0.2,5.1)

3.3 (1.0,ll.O)

5.8 [2.9,11.2] 2.6 [1.2,5.9] 2.2 [0.4,10.3]

4.1 [2.1,8.2] 0.8 [0.3,2.0] 1.2 [0.5,2.8]

1.0 (0.8,1.2)

3.0 (0.7,12.0)

(c) Nonhemorrhagic (thrombotic, embolic) stroke (ICD 432-438) 0.46 (39) 0.33 (31) 0.08 (11) RCGP (1983) Vessey et al. (1984) 0.34 (14) 0.10 (7) 0.13 (10) Ramcharan et al. (1981)f 0.53 (3) 0.29 (11) 0.24 (15) Fatal cerebral thrombosis, hemorrhage, or embolism (ICD 431-433) RCGP (1981b) 0.03 (2) 0.08 (8) 0.03 (4)

Standardization factors used in these three studies were listed in Section 111. ( ) Implies confidence interval given in reference cited; [ ] implies approximate confidence interval calculated using the methods described in Section 11. Excludes two cases of intracranial hemorrhage for which OC data not given, ICD 430,433,435-437 only. Significance level for x2 test of equality of relative risks. f ICD 433,435-437 only. a

b

302

ROSS L. PHENTICE A N D DAVID B. THOMAS

the standardization procedure for the Oxford cohort (Vessey et al., 1984) may represent “overstandardization,” and hence reduced relative risks. Specifically there would be little likelihood of important association between contraceptive choice and blood pressure prior to cohort selection (e.g. Vessey et ul., 1976, Table S ) , so that differences in subsequent hospital referrals for hypertension may arise from the effect of OC usage on blood pressure. The three studies are somewhat less consistent with respect to the implications of former OC use. The RCGP study identified a significantly elevated relative risk, estimated as 2.6, associated with former use, while the other two studies suggested small and nonsignificant increases. The homogeneity x’ test had significance level 0.11. The three studies combined give a summary estimated relative risk of 1.8 associated with former usage, with an approximate 95% confidence interval from 1.3 to 2.6. The issue of elevated relative risk following cessation of OC w e is of considerable importance. For example, the health consequences could be substantial if cerebrovascular disease relative risks remained noticeably elevated for many years following the cessation of OC use since the women would carry such elevated risks into the age range wherein these diseases become relatively common. Based on much smaller numbers of events the RCGP (1981a) report gives relative risks and confidence intervals for fatal cerebrovascular disease. Note that a significant elevated relative risk, estimated as 3.6, is observed in former OC users. Moreover, of the 19 cerebrovascular disease deaths among former OC users, only 1 experienced a (nonfatal) stroke while still taking oral contraceptives. Section (a) of Table 111 gives corresponding results for all cerebrovascular disease based on case-control studies, By far the largest such study was carried out by the Collaborative Group for the Study of Stroke in Young Women (1973, 1975). This study identified 598 nonpregnant women, 15-44 years of age, with various types of cerebrovascular disease, between 1969 and 1971. Each case was matched on age, sex, and race to both a hospital and a neighborhood control. Oral contraceptive histories were obtained by lay interviewers for about 70% of cases and controls. A relative, rather than the subject, provided information for about 12% of study subjects. The Collaborative Group reports emphasized current OC use and grouped together never and former usage into a “nonuser” comparison group. A highly significant estimated relative risk of 2.3 was obtained for current use of OCs by comparing cases with neighborhood controls. Comparison with hospital controls yielded an estimated relative risk of 2.6 with correspond-

TABLE I11 ORALCONTRACEPTIVES AND CEREBROVASCULAR DISEASE: CASE-CONTROL STUDIES Caseslcontrols Current users

Source

(a) All cerebrovascular disease Collaborative Group 123168

Former users

I

Neverusers

1

3071382

RR estimate (95% CI)

Combined RR estimate (95% CI)

Current users

Former users

Current users

Former users

1.4 [0.8,2.71 1.5 [1.0,2.31

1.0 [0.7,1.61 (p = 0.71)

1.5 [1.0,2.1] ( p = 0.48)

2.3 [1.6,3.1]n*b

(1973, 1975)

(b) Hemorrhagic stroke Collaborative Group

151/179

44/26

2.0 [1.2,3.41"

(1973, 1975)

Fatal subarachnoid hemorrhage Inman (1979) 33133 Thorogood et al. (1981) 27165 (c) Thrombotic stroke Collaborative Group

59114

48142c 64/94

I

1.3 [0.7,2.41 0.9 [0.5,1.61

32/40 591130

I

811138

7.2 [3.8,13.71

(1973, 1975)

Nonfatal thrombotic stroke Vessey and Doll (1969) Sartwell et al. (1969) Jick et al. ( 1978a)d Fatal thrombotic stroke Inman and Vessey

11131 811 1117

(1968)

51171 ~~~~~

6.1 [2.5,15.1] 19.2 [2.5,1491 25.7 [5.7,115.3]

81137 5112 3149

1.8 [0.7,4.9]

211827 ~

10.7 [5.4,21.3] ( p = 0.16)

~

~

Relative risk estimates and confidence intervals based on Table 111 of Collaborative Group (1973). [ ] Implies confidence intervals calculated using methods described in Section 11. All ever-users of OCs not known to be current users are regarded as former users. d Includes one case of nonfatal subarachnoid hemorrhage.

a

304

ROSS L. PRENTICX AND DAVID B. THOMAS

ing 95% confidence interval from 1.8 to 3.7. These estimates are quite consistent with, but somewhat less than, the combined relative risk estimate of 2.9 from the cohort studies. The inclusion of former users in the nonuser category tends to reduce the relative risk estimate. On the other hand, the relative risk estimate is likely somewhat inflated by a larger fraction of regular smokers in the case group (74%) as compared to the neighborhood (54%) or hospital (60%)control groups. Cases and control groups were, however, shown to be fairly comparable in respect to marital status, religion, education, income, and surgical sterilization of the subject or her husband. Consider now hemorrhagic stroke in relation to OC usage. Panel (b) of Table I1 gives results from the three large cohort studies for subarachnoid hemorrhage. Current OC usage is associated with somewhat, but nonsignificantly, elevated incidence in the two British studies and with very substantially elevated incidence in the Walnut Creek study. In spite of the considerable variation in the three estimated relative risks the number of events is small in all three studies, and no significant evidence of heterogeneity exists ( p = 0.13). The three studies yield a combined relative risk estimate of 2.0 for subarachnoid hemorrhage with an associated approximate 95% confidence interval of 1.1to 3.6. The relative risk estimates associated with former OC usage are quite similar for the three studies. The studies yield a summary relative risk estimate of 2.1 (approximate 95% confidence interval from 1.1 to 3.9), virtually identical to that for current OC use. A rather thorough reanalysis of the Walnut Creek subarachnoid hemorrhage data, using “case-control within cohort” methods (Petitti and Wingerd, 1978; Petitti et uZ., 1979), yielded significantly elevated relative risk estimates of 6.5 and 5.3 for current and former OC usage, respectively. The RCGP (1981a) report, based on a small number of deaths, exhibited elevated relative risk estimates for fatal subarachnoid hemorrhage of 3.2 and 4.5 for current and former OC usage, respectively, the latter of which was significantly greater than unity. Note that these estimates are somewhat larger than the corresponding incidence relative risks in the RCGP group. The RCGP (1983) report gives separate results for cerebral hemorrhage. As shown in Table I1 (b) former, but not current, use of OCs is associated with a significantly elevated relative risk of 3.3 based on a small number of cases. Hemorrhagic stroke categories were combined in the Collaborative Group for the Study of Stroke in Young Women (1973, 1975) casecontrol study report. An unmatched analysis based on 196 cases and 205 neighborhood controls (Table 111, part b) gives a relative risk

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

305

estimate of 2.0 for current OC use (compared to nonusers) with approximate 95% confidence interval from 1.2 to 3.4. Note that these estimates seem preferable to those given in the Collaborative Group’s 1975 report, which combined controls corresponding to both hemorrhagic and thrombotic cases. The corresponding relative risk estimate and confidence interval based on a comparison with hospital controls were very similar as were relative risk estimates from corresponding matched pair analyses. These estimates agree closely with the corresponding cohort estimates. One would expect somewhat larger relative risk estimates if current OC users were compared to never-users. Two additional case-control studies reported on OC use in relation to fatal subarachnoid hemorrhage. Inman (1979) reported on 134 women in the age range 15 to 44 who died in 1976 of subarachnoid hemorrhage along with an equal number of controls, pair-matched to the case on age, selected from the files of the general practitioner for the case. Cases and controls turned out to be similar with respect to marital status, and to parity as well. Cigarette smoking habits were not known for a majority of the sample. If, as an exercise, ever-users of oral contraceptives who were not known to be current users are taken to be former users one obtains relative risk estimates of 1.3 and 1.4 for current and former usage, respectively, neither of which is significantly greater than unity. Slightly larger relative risk estimates (1.4 for current use, 1.5 for ever-use) are obtained if the analysis is restricted to pairs in which neither member had a diagnosis of hypertension. Thorogood et al. (1981) reported on 158 women dying of subarachnoid hemorrhage during 1978, each of whom was matched on age and marital status to two control women identified from the files of the same general practitioner. Once again cigarette smoking habits were unavailable for a majority of study subjects. Relative risk estimates of 0.9 for current OC usage and 1.5 for former OC usage can be calculated from the published data; the latter value is nearly significantly greater than unity ( p = 0.06). The two studies combine to give borderline evidence of increased risk among former OC users but do not provide evidence for increased risk among current users. These case-control studies yield relative risk estimates for fatal subarachnoid hemorrhage that are noticeably less than the corresponding RCGP study estimates for both current and former use, though the confidence intervals are extremely wide in the RCGP study in view of the small numbers of deaths. The fact that the case-control relative risk estimates for fatal subarachnoid hemorrhage are less than the corresponding relative risks for disease incidence argues against the hypothesis in the RCGP (1983) report that OC use is associated with

306

ROSS L. PHENTICE AND DAVID B. THOMAS

an increased case fatality rate. The issue of relative risk for fatal subarachnoid hemorrhage appears to merit further study. Based on the data reviewed above it seems apparent that former OC use is associated with an increased relative risk. Perhaps a relative risk estimate in the vicinity of2.0 would be a reasonable guess at this point in time. Now consider the relationship between nonhemorrhagic (thrombotic) stroke and OC use. Part (c) of Table I1 shows relative risk estimates ranging from 2.2 to 5.8 for current OC usage from the three large cohort studies. Once again the relative risk estimates from the Oxford and Walnut Creek studies are lower than those from the RCGP study. Heterogeneity among the three relative risks is, however, not significant, and when combined they give a relative risk estimate for nonhemorrhagic stroke of 3.8 with corresponding approximate 95% confidence interval from 2.4 to 6.1. This relative risk then appears to be considerably larger than the corresponding hemorrhagic stroke relative risk. The three cohort studies yield quite varied relative risk estimates, ranging from 0.8 to 4.1 for former OC usage in relation to thrombotic stroke. The relative risk is significantly greater than unity in the RCGP study, but not in the other two. If combined, the three cohort studies yield a significantly elevated relative risk with point estimates of 1.9. Note, however, that there is significant heterogeneity ( p = 0.01) among the three relative risk estimates. The Collaborative Group for Study of Stroke in Young Women (1973) report (Table 111, c) gives a highly elevated thrombotic stroke relative risk estimate of 7.2 for current OC usage, which noticeably exceeds the summary relative risk estimate from the cohort studies. Three small case-control studies examined nonfatal thrombotic stroke in relation to current OC usage. As part of a larger study of thromboembolic disease, Vessey and Doll (1969) reported on the current OC usage of 19 patients, aged 16-40, with cerebral thrombosis, discharged from hospitals during 1964-1967, along with the OC usage for two hospital controls per case, matched on age, parity, date of admission, and absence of predisposing cause of thromboembolic disease. The study identified a significantly elevated relative risk associated with current OC usage, estimated to be 6.1. A similar study by Sartwell et al., (1969) included 13 cases, age 15-44, of nonfatal thrombotic stroke which were pair-matched to hospital controls on age, race, marital status, parity, absence of predisposing risk factors, and certain other factors. An unmatched analysis gives a relative risk estimate of 19.2 for current OC use, which is significantly greater than unity. In a more recent study Jick et al. (1978a) reported on 14 premenopausal

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

307

cases of nonfatal cerebrovascular disease (including one case of subarachnoid hemorrhage), each of whom was matched to four hospital controls on age, country of residence, and absence of predisposing medical conditions or contraindications to OC use. Current OC use was associated with a highly significant relative risk, estimated to be 25.7. When combined the three studies give a relative risk estimate of 10.7 for current OC use, with an approximate 95%confidence interval from 5.4 to 21.3. Thus nonfatal thrombotic stroke may be of particularly elevated incidence in relation to current OC usage. The extent to which such estimates may be inflated by closer medical surveillance of OC users, by the exclusion of predisposing conditions, or by residual confounding (e.g., by cigarette smoking habits) is unclear. Inman and Vessey (1968) included in a broader study 26 cases of fatal thrombotic stroke, aged 20-44, along with controls selected from the records of the case’s general practitioner. Comparison to the total control group for the study yields a relative risk estimate of 1.8 for current OC use. The elevation in risk is not significant at the 0.05 level. When cases with a predisposition to stroke were excluded, however, the estimated relative risk increased to 5.7 with an approximate 95% confidence interval from 1.9 to 17.3. The RCGP (1981a) report does not identify fatal cerebral thrombosis separately. As shown in the final row of Table 11, however, fatal cerebral thrombosis, hemorrhage, or embolism has relative risk estimates of 1.0 and 3.0 for current and former use of OCs. The analysis is based on extremely small numbers, however, and neither relative risk is significantly greater than unity. In summary, current use of OCs is associated with an estimated relative risk of perhaps 4 or 5 for thrombotic stroke. This relative risk may be larger for nonfatal than fatal stroke and larger if only women without a predisposition to stroke are studied than if all women are studied, though the literature is not definitive on these points. The latter difference may simply reflect some avoidance of OCs among women with a predisposition to stroke. The literature is also not clear in respect to the thrombotic stroke relative risk associate with former OC use. There does appear to be some increased risk, however, with relative risk perhaps in the vicinity of 2.0.

2 . Dependence of Cerebrovascular Disease Relative Risks on OC Dose and Formulation, Duration of Use, Time since Cessation of Use, and on Other Factors The cerebrovascular disease relative risks described above, for current and former OC usage, do not address a number of key issues. For example, the relative risk associated with current use may well de-

308

ROSS L. PRENTICE AND DAVII) H. THOMAS

pend on such aspects of the preceding OC history as duration of use, time since first use, and regularity of use since first usage. Similarly the relative risk may depend on the dosage and potency of both the estrogen and the progestogen in the combined OC pill and perhaps on the relative potency between the two. The same issues are pertinent to the relative risk associated with former OC usage., while the dependence of the relative risk on time since cessation of use is of considerable additional importance. Finally it is of interest to examine the dependence of relative risks on such disease risk factors as age, cigarette smoking habits, and blood pressure levels. Most of the studies cited above have not been in a position to examine explicitly detailed aspects of OC formulation and usage pattern in relation to cerebrovascular disease relative risk, either because of a modest number of disease events or because of a lack of detail in available OC usage data. As shown in Table IV, however, the RCGP cohort (RCGP, 1983; Kay, 1982) has yielded some valuable insights on these topics. The relative risk for total cerebrovascular disease in the RCGP study is not significantly dependent on duration of use among current users, though a positive trend is suggested. Similarly (Table IV) the relative risk among former users appears to be fairly constant as a function of time since cessation of use, for more than a 6-year period. One might speculate that there may be some confounding by OC dose and formulation in this table, with longer durations perhaps more likely to correspond to higher doses. Kay (1982) studied the role of progestogen dose, based on the same database used in RCGP (1983). It happened that 25 of the 51 cerebrovascular disease events among current users (Table 11, a) involved three OC preparations, formulated by a single manufacturer, with 50 pg of ethinyl estradiol (an estrogen) and varying doses (1,3,or 4 mg) of the progestogen norethindrone acetate (NEA). As shown in Table V, the relative risk increases significantly ( p < 0.05), and apparently approximately linearly, with increasing NEA dose. The combined experience with these preparations gives a relative risk estimate (3.1)identical to that for the RCGP cohort as a whole, whereas the estimated relative risk corresponding to the lowest of the three progestogen doses is estimated to be 2.0. The corresponding (approximate) 95% confidence interval [0.8,4.9] is, however, very broad. Valuable support for the importance of the progestogen component in relation to cerebrovascular disease incidence is provided by a study (Meade et al., 1980) based on reports to the Committee on Safety of Medicine in the United Kingdom. A total of 191 occurrences of stroke were reported among current users of the same three preparations

TABLE IV DEPENDENCE OF CEREBROVASCULAR DISEASE RELATIVERISK (RR) ON DURATION OF OC USE AMONG CURRENTUSERS, AND ON TIMESINCE CESSATION OF OC USE AMONG FORMER USERSO Total duration (in months) of OC use (current users)

1-24

25-48

49-72

73-96

0.37 (8) 1.9 [0.9,4.31

0.49 (10) 2.6 [1.2,5.31

0.78 (14) 4.1 [2.1,7.91

0.81 (11) 4.3 [2.1,8.61

97 +

Never users

~~

Cases per 1000 person-years (number of cases)b Estimated relative risk 95% CI for RR"

0.51 (8) 2.7 [1.2,5.9]

0.19 (26)

Months since last OC use (former users)

Cases per 1000 person-years (number of cases)d Estimated relative risk 95% CI for RRc

1-6

7-12

13-24

25-48

0.69 (6) 3.5 [1.4,8.41

0.56 (4) 2.8 [1.0,8.01

0.39 (5)

0.44 (9) 2.2 [1.0,4.71

1.9

[0.7,5.11

Based on RCGP (1983). Standardized for age, parity, sociai class, and smoking habits at recruitment. Calculated by approximate methods described in Section 11. Standardized for age.

49-72 0.78 (11) 3.8 [1.9,7.91

73+ 0.78 (11) 3.8 [1.9,7.91

Never users

0.20 (26)

3 10

HOSS L. PHENTICE AND DAVID €3. THOMAS

TABLE V DEPENDENCE OF CEREBROVASCULAR DISEASE RELATIVERISK( R R ) ON DOSAGE OF THE PROGESTOGEN NORETHINDRONE ACETATE(NEA) AMONG oc PREPARATIONS WITH A COMMON ESTROGEN COMPONENT‘

Ethinyl estradiol (50 p g ) and NEA at

Cases per 1OOO person-years6 (number of cases) Estimated relative risk 95% CIc for RR

1 mg

3 mg

4 mg

Neverusers

0.38 ( 6 )

0.72 (14)

1.25 (5)

0.19 (26)

2.0 [0.8,4.9]

[2.0,7.3]

3.8

6.6 [2.5,17.1]

“ From Kay (1982). Standardized for age and parity at diagnosis and smoking habits at recruitment. Using approximate methods described in Section 11.

mentioned in the preceding paragraph. A significant positive trend (p < 0.01) was detected between the ratio of observed to expected stroke occurrence and the NEA dosage, with expected rates calculated on the basis of retail pharmacy purchase figures. At face value these data would suggest relative risk estimates of 1.5 and 1.9 for the 3 and 4 mg preparations compared to the 1 mg preparation. These “estimates” are consistent with, but somewhat smaller than, the corresponding ratios from Table V. Meade et al. (1980) also reported a significantly higher observed to expected stroke occurrence among current users of preparations with a higher dose (0.25mg) of the progestogen levonorgestrel as compared to a lower dose (0.15 mg), with both preparations having 30 p g of ethinyl estradiol. In addition, the two stroke events among current users of these two preparations in the RCGP study (Kay, 1982) occurred at the higher dose. Meade et ul. (1980)also attempted to relate cerebrovascular disease to estrogen dose. They reported somewhat lower stroke occurrence (p > 0.10) at 30 as compared to 50 pg of estradiol, as had been suggested in an earlier report (Inman et al., 1970).This information, however, seems virtually impossible to interpret since differences in corresponding progestogen doses and potencies were not acknowledged. The progestogen dose-response relationship, the importance of the estrogen component, and the importance of the relative potencies of the two steroids in relation to stroke occurrence are key topics for further study. A number of groups have reported on the dependence of the cerebrovascular disease relative risk on other risk factors. For example, Table VI, from the Collaborative Group for the Study of Stroke in

TABLE VI DEPENDENCE OF CEREBROVASCULAR DISEASE RELATIVE RISK(RR) ASSOCIATED WITH CURRENT OC USE ON CIGARETTESMOKING HA BITS^ Smoking habits Now regular

Never regular

Hemorrhagic stroke Caseslcontrolsb Relative risk 95% CI for RR" Thrombotic stroke Caseslcontrols Relative risk 95% C I for RR

b c

Once regular

(< 1 packlday)

Now regular ( 2 1 pack/day)

User

Nonuser

User

Nonuser

User

Nonuser

User

Nonuser

6/31 1.0 [0.4,2.5]

351176

7/12 1.2 [0.4,3.51

19/39

9/11 1.6 [0.6,4.21

38/74

21/15 2.3 [ l .1,4.8]

57/93

18/31 3.3 [1.6,6.6]

31/176

13/12 5.3 [1.8~5.81

8/39

10111 3.7 [ 1.4,10.2]

18/74

18/15 4.6 [2.0,10.5]

24/93

Based on Collaborative Group (1975). Neighbor controls. Using approximate methods described in Section 11.

312

ROSS L. PRENTICE AND DAVID B. THOMAS

Young Women (1975) report, indicates the relative risk associated with current OC use (based on comparison with neighborhood controls) to be fairly constant across cigarette smoking categories for both hemorrhagic and thrombotic stroke. If anything, the OC relative risks are higher among former and current smokers than among never smokers. Thus, in view of a noteworthy stroke risk associated with cigarette smoking itself (particularly hemorrhagic stroke), the excess risk associated with OC use is particularly large among current and former smokers. The RCGP (1983) report gives a display of relative risk as a function of current or former OC use, smoking status, and age category, but only for total arterial diseases combined. Relative risks associated with current or former OC use were generally similar among smokers and nonsmokers, but relative risks appeared to be somewhat higher among older (age 35+) than among younger women (aged ~35). The Collaborative Group (1975) report also examined the dependence of cerebrovascular disease relative risk associated with current OC use on blood pressure by comparing maximal recorded blood pressures for cases and hospital controls. Relative risks for both hemorrhagic and thrombotic stroke associated with OC use varied little across normal blood pressure, mild, moderate, and severe hypertension groups, with perhaps smaller relative risks among severe hypertensives. Any such reduction is difficult to interpret since OC use likely contributes to some hypertension diagnosis and since severe hypertension may have been regarded as a contraindication to OC use for some subjects. In summary, the relative risks for hemorrhagic or thrombotic stroke associated with OC use seem to be rather similar among women with and without disease risk factors. A simple multiplicative relative risk model appears to be reasonably in line with the collective data.

B. ISCHEMIC HEARTDISEASE 1 . Relatiue Risks Associated with Current and Former OC Use Table VII summarizes ischemic heart disease incidence and mortality by OC use category for the three previously described cohort studies. Part (a) shows incidence rates, standardized as described in Section I11 for total ischemic heart disease. Apparently such data have not been reported from the Oxford Family Planning Association cohort. The RCGP and Walnut Creek cohorts both gave relative risk estimates of 1.4 associated with current OC use, providing evidence for an in-

TABLE VII ORALCONTRACEPTIVES AND ISCHEMIC HEARTDISEASE: COHORT STUDIES ~

Cases per 1000 person-years (number of cases) Current users

Source

-

b

Former users

Neverusers

RR estimate (95% GI)"

Combined RR estimate (95% CI)

Current users

Current users

Former users

~~

[ ] Implies confidence interval calculated using the approximate methods described in Section 11. Of the eight ever-users of OCs, two were OC users at the time of death and six were former users.

Former users

3 14

ROSS L. PRENTICE AND DAVID H . TtiOhlAS

crease that is just significant at the 5% level. The corresponding relative risk among former OC users are both slightly greater than one, giving a combined estimate of 1.2, which is not significantly greater than unity. The two British cohort studies have reported data on fatal ischemic heart disease. The RCGP study gives an increased relative risk estimate (6.4) for current OC use, with a nonsignificant relative risk estimate of 2.0, based on a very small number of deaths, among former OC users. The Oxford Family Planning study provides a large, but nonsignificant, relative risk estimate (5.7) for ever-use of OCs, based on a small number of deaths. When attention is focused on myocardial infarction (MI) incidence (Table VII, b) the RCGP study gives a relative risk estimate of 2.0 ( p < 0.05) for current OC use, as compared to 1.1 from the Walnut Creek study. There is no evidence of heterogeneity between the two, and evidence for an increase remains (p < 0.05) when the two studies are combined. There is no evidence for an increased M I incidence among former users, the combined study relative risk estimate being only 1.1. The Walnut Creek data have been analyzed by Petitti et nl. (1979) using case-control within cohort regression methods. These analyses agree with those of Ramcharan e t ul. (1981). Specifically, they produced myocardial infarction relative risk estimates of 0.8 for current and former O C use, with corresponding 95% confidence intervals of (0.2,2.6)and (0.4,1.7),respectively. The final part of Table VII (c) examines the rates of ischemic heart disease exclusive of (concurrent or subsequent) myocardial infarction. The RCGP and Walnut Creek studies combine to give relative risk estimates of 1.3 for current and 1.2 for former OC use, neither of which are significantly greater than unity. In summary, the cohort studies provide evidence for some increase in ischemic heart disease among current OC users, while suggesting that any elevated incidence among former users may be modest. The relative risk for myocardial infarction associated with current OC use may be particularly elevated, though it is not estimated with precision based on these cohort studies. A rather large number of case-control studies have attempted to estimate the relative risk of myocardial infarction among OC users. Part (a) of Table VIII gives results for nonfatal myocardial infarction from eight case-control studies. Vessey and Doll (1969) included 17 married women discharged during 1964-1967 following an MI in a study of OCs and a range of thromboembolic diseases. Cases were matched to two controls on age, parity, and date of admission, and both groups were without predisposing factors for thromboembolic

TABLE VIII AND ISCHEMIC HEARTDISEASE: CASE-CONTROL STUDIES ORALCONTRACEPTIVES

(b) Fatal myocardial infarction Inman and Vessey (1968) Mann and Inman-(1975) Krueger et al. (1980)e Adam et al. (1981a)

231171 41124 12134 24/38

1711827 1 18125 1401182 I 631292 1 35170 641137

I

1.0 r0.6.1.51 2.2ii.313.83 0.9[0.5,i.81 1.6(0.7,3.4) 1.4(0.7,2.4) 1.1 [0.6,1.8]

1.4[1.0,1.91 1.0[0.7,1.5] ( p = 0.16) (p = 0.75) ~

[ ]Implies confidence interval calculated using approximate methods described in Section 11. Predisposed cases and controls only. Excludes study of Jick et al. (1978b). Idiopathic cases and controls only. Includes “definite” MI cases only.

316

ROSS L. PRENTICE AND DAVID B. T H O M A S

disease. No association between MI and current OC use was suggested. Cigarette smoking data were collected but were not used in RR estimation. Stolley et al. (1975) also included a small number (28) of MI cases in a more general hospital-based case-control study of thrombosis among women aged 15-49. They reported an overall MI relative risk estimate of 1.9 for current OC use and a relative risk estimate of 7.2 among idiopathic (nonpredisposed) subjects. Cases and controls were initially matched on age, race, marital status, hospital, and date of admission. Further analysis (Maguire et al., 1979) introduced additional matching according to predisposition to thromboembolic disease. A range of additional important confounding factors, including cigarette smoking, cholesterol, hypertension, and diabetes, were also controlled using logistic regression methods. The resulting myocardial infarction relative risk estimate for predisposed cases was only 1.3, as is given in Table VIII, while idiopathic cases were too few to permit such an analysis. Mann et al. (1975a,b) reported on a small case-control study of MI among women less than 45 years of age. Three control women with other diagnoses were matched to each case on marital status, age, and year of admission. The relative risk estimates of4.3 and 1.1 for current and former OC use were obtained without further standardization. The authors comment, however, that the R R estimate for current use is reduced to 3.1 after standardization for hypertension, preeclamptic toxemia, cigarette, and hypercholesterolemia, and this number is included in Table VIII. The small hospital-based case-control report by Rosenberg and Armstrong (1976) involved premenopausal women in seven countries diagnosed after 1969 and gave a RR estimate of 1.3 for current OC use. The estimate was standardized for cigarette smoking, history of hypertension, angina, or diabetes, age, and country. Arthes and Masi (1976) reported on 65 predisposed MI cases, aged 30-44, and 254 controls without chronic disease or conditions related to thromboembolism, admitted to 1 or 47 U.S. hospitals. Their data give an age-adjusted RR estimate of 1.8 for current OC use. The results of Jick et al. (1978b) stand as an outlier in Table VIII. The results shown arise from 26 idiopathic MI cases discharged during 1975 from 1 of 621 hospitals. These cases and age- and hospitalmatched controls give rise to a relative risk estimate of m.7 for current and 3.6 for former OC use. Such inconsistency with the other studies listed may well arise from extreme selection; in fact, the 26 M I cases arose from a total of 954 patients with a discharge diagnosis of MI. The corresponding report of predisposed XI1 (Jick et al., 1978c) included no controls who were OC users. These studies are excluded from the heterogeneity test and combined study RR estimation in Table VIII.

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

317

Shapiro et al. (1979) and Slone et al. (1981) reported on a substantial study including 556 premenopausal women (ages 25-49) admitted to the coronary care units of 155 hospitals in Boston, New York, or Delaware Valley during 1976-1978. A total of 2,036 controls were selected from the same or a nearby hospital, with diagnosis judged to be unrelated to OC use. Relative risk estimates of 3.3 and 1.1were obtained for current and former OC use. The relative risk estimate for current OC use is highly significant and is adjusted via logistic regression for age, ethnic group, cigarette smoking, weight/height2, diabetes, lipid abnormality, hypertension, angina pectoris, preeclamptic toxemia, and hospital. The study by Rosenberg et al. (1980) is unique among those listed, in that it makes use of healthy control women. Cases and controls were among 121,964 U.S. nurses responding to a mail questionnaire. The 156 premenopausal women who reported hospitalization prior to the menopause for MI were matched on age and menopausal status (at time of MI) to other respondents. Relative risk estimates of 1.8and 0.9 were obtained for current and former OC use, respectively. Relative risk estimates were adjusted via logistic regression for obesity, current cigarette smoking, history of hypertension, elevated cholesterol, diabetes, or angina pectoris, or parental MI before age 50. AS shown in the right-hand columns of Table VIII the seven casecontrol studies (exclusive of Jick et d.,1978b) of nonfatal myocardial infarction combine to give a relative risk estimate of 2.2 for current OC use with a corresponding 95% CI of [1.6,2.9]. This estimate is in excellent agreement with that (1.9) from the cohort studies but is noticeably less than the 3- to 4-fold increase commonly referred to in the literature (e.g., Rosenberg et al., 1980).There is no evidence of heterogeneity ( p = 0.35) among the relative risks from the contributing studies. The three studies that report on former OC use give rise to a nonsignificant RR estimate of 1.1,identical to that from the cohort studies. The two sources combined yield an approximate 95% CI of (0.9,1.3) so that the issue of some small increased MI risk among former users is not settled by this analysis. Panel (b)of Table VIII describes aspects of four case-control studies of fatal myocardial infarction. Inman and Vessey (1968) obtained U.K. death certificate transcripts mentioning thrombosis or embolism of coronary vessels among women aged 20-44, Attempts were made to interview the general practitioners who cared for each deceased case, and control women were selected from the general practitioner’s files. This study gives an age- and parity-adjusted relative risk estimate of 1.0 for current OC use upon bringing in the control group for the entire case series, which also included as cases pulmonary and cere-

3 18

ROSS L. PRENTICE AND 1)AVID B. THOMAS

bra1 thrombosis and embolism. Mann and co-workers (Mann and Inman, 1975; Mann et al., 1976) reported on a study of similar design, restricted to myocardial infarction. This study gives crude relative risk estimates of 2.2 and 0.9 for current and former OC use, respectively. None of the studies just mentioned had adequate cigarette smoking data. Cigarette smoking data were, however, obtained in the study of Krueger et al. (1980). Computer files of the U.S. National Center for Health Statistics were searched for women aged 15-44 who died of acute MI in 1974-1975 in five large Standard Metropolitan Statistical Areas. Both deceased and hospitalized but surviving controls were matched to cases on age, geographic area, and year of death or hospitalization, and next-of-kin or closest relative/friend interviews were conducted to collect information on OC use, cigarette smoking habits, and other factors. This study gave an age-adjusted relative risk estimate of 1.6 for current OC use. This estimate was not much affected by controlling for cigarette smoking habits. Finally Adam et al. (1Y8la) reported a further death certificate-based study with cases being women aged 15-44 dying of MI in England and Wales in 1978. Controls matched on age and marital status were selected from the records of the case’s general practitioner. Relative risk estimates of 1.4 and 1.1 were obtained for current and former OC use. Somewhat larger estimates were obtained for nonpredisposed women. Cigarette smoking information was not considered. These studies combine to give summary fatal MI relative risk estimates of 1.4 and 1.0 for current and former OC use. As mentioned above data from the early study by Innian and Vessey (1968) were not presented in a manner that permits well-controlled RR estimation. Its exclusion improves the degree of homogeneity among RR estimates ( p = 0.48) and leads to a summary RR estimate of 1.8 with 95% CI of [1.2,2.6] in quite good agreement with the nonfatal MI results. In summary, current use of OCs is associated with an approximate 2-fold increase in MI incidence based on a substantial series of studies that generally provide accommodation for such MI risk factors as cigarette smoking, elevated serum cholesterol, and elevated blood pressure. Control of these factors is essential since they have evidently played a role in decisions regarding prescription of contraceptives. On the other hand, the adjustment procedures used do not acknowledge the impact of OC use on blood pressure or on plasma lipids or lipoproteins. Hence the procedures employed may represent some overcorrection, with resulting deflation of the estimated relative risks. Aside from the point just made, the above analyses suggest that any overall increased MI risk among former OC users must be small.

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

319

2 . Dependence of Ischemic Heart Disease Relative Risks on OC Dose and Formulation, Duration of Use, Time since Cessation of Use, and on Other Factors

A few of the studies described above have examined the dependence of the relative risk associated with current OC use on duration of use. Part (a) of Table IX shows such results for all ischemic heart disease from the RCGP (1983) study. No trend with duration of use is apparent over 8 years or more of exposure. Part (b) of Table IX shows the dependence of the nonfatal MI relative risk on duration of exposure for current OC users in the case-control studies reported in Rosenberg et al. (1980) and Slone et al. (1981). Both studies give relative risk estimates (4.4)that are larger for women with 10 years or more of exposure than for women with shorter term usage. Neither study, however, yields a significant trend. When the two studies are combined (as indicated in Table IX footnotes), however, the relative risk is shown to increase with increasing duration of exposure. For example, the RR among women with 10 or more years of OC exposure is significantly greater ( p < 0.05) than that among women with less than 5 years of exposure. Slone et al. (1981) also examined the dependence of the RR on duration of use among former OC users (Table IX,c). The relative risk was shown to increase significantly with increasing duration of OC use. The authors further examined the relationship of the RR to time since last use of OCs among former users with 5 or more years of OC use. No trend was apparent though an increased risk was evident as long as 5-10 years after discontinuation of use. These results need to be interpreted cautiously in view of the failure to show an overall increased MI risk among former OC users (Tables VII and VIII; Layde et al., 1982b). The data cannot be said to be strong with respect to the importance of duration of OC use among current users and concerning the duration of any elevation in risk among former users. These topics are of enormous practical importance, and further examination in one or more large-scale studies is merited. As shown in Table X the report of Kay (1982) suggests an increasing ischemic heart disease RR (among current OC users) with increasing dose of the progestogen norethindrone acetate (NEA) at 50 pg of ethinyl estradiol, though the trend is not significant. The relative risk estimates of 2.2 and 1.9 for the 3 and 4 mg doses of NEA compared to the 1mg dose, however, agree well with the corresponding observed to expected ratios of 2.2 and 2.2 in the report of Meade et al. (1980).As described previously, these latter ratios are based on reports to the

TABLE IX DEPENDENCE OF ISCHEMICHEARTDISEASE RELATIVERISKON DURATION OF OC USE AMONG CURRENT AND FORMER OC USERS Total duration (in months) of OC use w

s

25-48

49-72

73-96

97+

Neverusers

0.93 (12)

0.64 (10)

0.64 (10)

0.66 (9)

0.70 (13)

0.49 (75)

1.9 [1.0,3.5]

1.3 [0.7,2.5]

1.3 [0.7,2.5]

1.3 [0.7,2.7]

1.4 [0.8,2.61

1-24 (a) All ischemic heart disease among current OC users RCGP (1983): Cases per 1000 person-years (number of cases) Estimated relative risk" 95% CI for RR*

Total duration (in years) of OC use

<1

(b) Nonfatal myocardial infarction among current OC users 4131 Rosenberg et al. (1980): Cases/controls Estimated relative risk 95% CI for RR Slone et al. (1981): Caseslcontrols

1-4

51107 0.7 2.7 [0.9,7.8] [0.3,1.8] I 10119 1

5-9

10+

71127 1.3 [0.6,2.9] 16132

7139 4.4 [1.9,10.11 14/27

Neverusers

9512036

30911223

40413259

<5 (c) Nonfatal myocardial infarction among former OC users 1281538 Slone et al. (1981): Caseslcontrols Estimated relative riskscl 1.0 95% CI for RR [0.7,1.21

10+

411127 1.4 [l.0,2.01

28/48 2.3 [1.5,3.81 ~

0

2 !

Neverusers

5-9

~

30911223

~~

Standardized for age, parity, social class, and smoking status. Confidence intervals throughout the table were obtained using the simple approximations described in Section 11. RR estimates stratified on age. Log-relative risk estimates for combined categories or studies obtained as a variance weighted linear combination of individual log-relative risk estimates. Age categories combined in the manner described in footnote d.

322

ROSS L. PHENTICE AND DAVID B. THOMAS

TABLE X DEPENDENCE OF ISCHEMICHEART DISEASE RELATIVERISKON DOSAGE OF NORETHINDRONE ACETATE (SEA) AMONG PHEPARKTIONS U'ITH A COMhlON ESTROGEN COMPONENT"

oc

Ethinyl estradiol (50 k g ) and NEA at lmg Cases per 1000 person-yearsb(number ofcases) Estimated relative risk 95% CI' for R R

3mg

4mg

0.40 (5) 0.87 (15) 0.75 (3) 0.8 1.8 1.5 [0.3,2.01 [1.0,3.01 [0.5,4.91

Neverusers 0.49 (75)

From Kay (1982).

* Standardized for age, parity at diagnosis, and smoking habits at recruitment. Using approximate methods described in Section 11.

U.K. Committee on the Safety of Medicines. The trend in the report of Meade et aE. was highly significant ( p < 0.001). All but 7 of the 120 ischemic heart disease reports in this analysis were reports of myocardial infarction. It then appears that progestogen dose is an important ingredient of the elevated A41 risk associated with oral contraceptives, with the incidence at 1 mg KEA being approximately one-half that at 3 or 4 mg. Several of the case-control studies cited above (e.g., Stolley et aE., 1975; Adam et al., 1981a) as well as Meade et al. (1980) and Inman et al. (1970) attempted to describe the importance of the estrogenic dose in the OC formulation. These reports tend to suggest higher MI incidence at greater doses of estrogen, but such an association is very likely confounded by progestogen potency in these studies. Some ofthe studies cited above also examine the dependence of the M I relative risk associated with OC use on other study subject characteristics or exposures. For example, several of the case-control studies (Table VIII) consider separately women that are predisposed to MI on the basis of such f'actors as hypertension, elevated serum cholesterol, diabetes, angina pectoris, preeclamptic toxemia, or family history of MI. Relative risk estimates tended to be somewhat higher among noiipredisposed women than overall, suggesting a relative risk model that is submultiplicative. For example, the overall RR associated with current OC use in Rosenberg et al. (1980) increases from 1.8 to 2.8, with corresponding 95% confidence interval [ 1.0,7.8], when the analysis was restricted to idiopathic (nonpredisposed) MI occurrence. The RE in Adam et al. (1981a) increases from 1.4 to 2.2 for current OC use, with 95% CI of [1.2,4.3] with such restriction, while that for former OC use increases from 1.1to 1.4 [0.8,2.6]. It is unclear whether such higher values represent a real difference or whether the lower values

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

323

among predisposed women arise from lack of adequate confounding factor control, as may arise if predisposing conditions tend to be more severe among OC nonusers than among OC users, or from a lack of acknowledgment of the impact of OC use on such predisposing factors as hypertension and hypercholesterolemia. The dependence of the MI relative risks for current OC use on cigarette smoking habits have been much studied. Maguire et al. (1979) did not find a significance dependence of the RR associated with OC use on smoking habits (1 month prior to interviews) though the estimate was somewhat smaller among smokers than among nonsmokers. The data of Mann et al. (1975a),however, yield RR estimates of 2.0 [0.5,8.6] and 5.2 [1.8,15.1] among nonsmokers and smokers, respectively, based on small numbers. In contrast Rosenberg et al. (1980) calculate a relative risk of 2.8 (1.0,7.8)among nonsmokers and 1.1 (0.5,2.6)among smokers, with both calculations restricted to nonpredisposed women. Shapiro et al. (1979) report RR estimates of 4.5 (1.4,14.1), 1.2 (0.3,4.4),and 4.3 (2.2,8.2) for current OC use among nonsmokers, smokers of 1-24 cigarettes per day, and smokers of 25 or more cigarettes per day, respectively. Finally Krueger et al. (1980) calculate a fatal MI relative risk of 2.2 (0.6,7.3)for current OC use among nonsmokers, as compared to an RR estimate that can be calculated to be 0.9 [0.4,1.7] among smokers. These estimates appear to suggest relative risks associated with OC use that are similar, but perhaps somewhat smaller, among smokers than among nonsmokers. Finally, certain of the case-control studies listed in Table VIII present separate relative risk estimates for current OC use by age category. For example, Slone et al. (1981) report relative risks of 3.6 [1.4,9.0],2.5 [1.2,5.0],and 5.9 [2.3,15.4] for age groups 25-34, 35-44, and 45-49, respectively, while other authors report slightly higher or slightly lower relative risks among younger as compared to older women. More generally, a simple multiplicative relative risk model involving OC use, cigarette smoking, and age seems a reasonable approximation. Under such a model older women who smoke have an absolute MI risk that is much increased by OC use and, if unable to stop smoking, are well advised to avoid OC use for this reason alone, as has been widely recommended. The reader is referred to Kay (1984) for a display of estimated excess circulatory disease mortality risks among OC ever-users, as a function of age and cigarette smoking habits. In addition to demonstrating a very substantial excess risk among older women who smoke, this display provides a useful reminder of the enormous effect that cigarette smoking has on arterial disease risk.

324

ROSS L. PRENTICE AND DAVID B. THOMAS

C. PERIPHERAL VASCULAR DISEASE

1 . Re1atit.e Risks Associated with Current and Former OC Use Table XI summarizes the relationship of both peripheral arterial and peripheral venous disease to OC use, based on the three previously described cohort studies. Part (a) of Table XI indicates a somewhat elevated risk of peripheral arterial disease among current OC users (summary relative risk estimate of 1.6)with no evidence for such an increase among former OC users. Most of the information on this disease category comes from the RCGP (1983) report, which excluded chilblains for which the relative risk and 95% confidence interval for current and former users were 1.5 (1.2,l.g)and 1.1 (0.9,1.4),respectively, based on a large number (418) of reported cases. Within the peripheral arterial disease category, the RCGP (1983) report showed significant increases for Raynaud's disease (ICD 443.0) and for arterial embolism and thrombosis (ICD 444), the latter based on a small number of cases. Venous thromboembolism was rather commonly reported in the three cohort studies. Most papers on this topic tended to emphasize idiopathic disease since the venous thromboembolism that is precipitated by surgical operations or severe trauma, for example, may relate rather differently to OC use than does that arising more spontaneously. Similarly women with certain predispositions to thromboemboiism (e.g., varicose veins) were often excluded from the analysis. Perhaps most importantly women with a previous history of thromboembolism were mostly excluded from the group eligible for nonpredisposed venous thromboembolism. Part (b) of Table XI gives results for such idiopathic thromboembolism with rates standardized within each cohort in the manner previously described (Section 11). Significant heterogeneity ( p = 0.04) can be detected among the three RR estimates associated with current OC use. Specifically the R R estimate for the RCGP cohort is considerably smaller than that from the Oxford (Vessey, 1978) and Walnut Creek (Petitti et al., 1978) cohorts. The relative risk estimate of 6.2 from the Oxford study arises from excluding women with a history of venous thromboembolism or varicose veins at admission to the study, and from excluding venous thromboembolism events that are associated with surgery. The 7.7 estimate from the Walnut Creek cohort arises by excluding disease associated with surgery, trauma, malignancy, pregnancy, or the puerperium and by excluding subjects with a range of predisposing factors (varicose veins, high blood pressure, history of thromboembolism, or

TABLE XI ORALCONTRACEPTIVES AND PERIPHERAL VASCULAR DISEASE: COHORTSTUDIES Cases per 1000 person-years Source

Current users

Former users

Neverusers

(a) Peripheral arterial disease (ICD 440-448) 1.31 (118) 0.90 (73) 0.79 (107) RCGP (1983)b 0.76 (5) 0.24 (9) 0.24 (15) Ramcharan et al. (1981)c (b) Idiopathic venous thromboembolism (ICD 450-453) 3.16 (115) 1.17 (16) 1.15 (53) RCGP (1978) 0.14 (6) I 0.87 (28) I Vessey (1978) Petitti et al. (1978) - (9) - (8) (c) Idiopathic pulmonary embolism (ICD 450) 0.19 (6) 0.08 (1) 0.08 (4) RCGP (1978) (d) Other idiopathic venous thromboembolism (ICD 451-453) 2.97 (109) 1.09 (15) 1.07 (49) RCGP (1978) (1

b c

RR estimate (95% CI)"

Combined RR estimate (95% CI)

Current users

Former users

Current users

Former users

1.6 (1.3,2.1) 3.2 (0.7,12.7)

1.1(0.8,1.5) 1.0 (0.5,2.7)

1.6 [1.3,2.1] (p = 0.35)

1.1[0.8,1.5] (p = 0.84)

2.7 [2.0,3.8] 6.2 [2.6,15.0] 7.7 (2.9,20.7)

1.0 [0.6,1.8]

3.2 [2.4,4.3] (p = 0.04)

2.4 [0.7,8.41

1.0 [0.1,8.91

2.8 [2.0,3.91

1.0 [0.6,1.81

~

[ ] Implies approximate confidence interval calculated using the methods described in Section 11.

Excludes chilblains (ICD 443.2). Excludes diseases of the capillaries (ICD 448).

326

ROSS L. PRENTICE AND DAVID B. THOMAS

diabetes). The 2.7 estimate from the RCGP cohort also excluded women with a range of predisposing factors and excluded women from the “risk set” close in time to surgical procedures or pregnancy. The RCGP (1978) report provides no evidence of increased venous thromboemholism risk among former OC users. Venous thromboembolism has proven fatal for a small number of women in these cohorts. The RCGP (1981a) report lists five deaths in this category, all of which involved pulmonary embolism. Three of the deaths occurred in current OC users, while the other two occurred in former OC users. One death in the Walnut Creek Study (Ramcharan et al., 1981) was attributed to pulmonary embolism, also an OC (ever) user. As of the time of the report of Vessey et al. (1981a),no deaths in this category has arisen in the Oxford Study. The remainder of Table XI indicates that the relative risks for the incidence of “idiopathic” pulmonary embolism in current and former OC users do not appear different from corresponding relative risks for other idiopathic venous thromboembolism (primarily phlebitis and thrombophlebitis), based on RCGP (1978). Table XI1 gives a more precise look at the OC relative risks for both idiopathic and predisposed thromboembolism, based on case-control studies. All of the case-control studies emphasized current, but not former, OC use. Hence the format of Table XI1 differs slightly from preceding tables. Most of the studies listed in Table XI1 have been mentioned in the preceding subsections. Vessey and Doll (1969) excluded disease associated with surgery or trauma or associated with a range of coexistent diseases. They also excluded superficial thrombophlebitis from their case group. Sartwell et al. (1969) (see also Sartwell, 1971) restricted their analysis to disease without associated medical, surgical, or traumatic condition, the medical conditions including all forms of intervascular thrombosis. The Boston Collaborative Drug Surveillance Program (1973)report was based on cases and hospital controls discharged from 1of 24 participating hospitals in the greater Boston area. Women were excluded with a past history of venous thromboembolism, varicose veins, or for other reasons that indicate predisposition, lack of need for contraception, or contraindication to OC use. The RR estimate reported (11.0)is standardized only for age. The study of Stolley et al. (1975) and Maguire et al. (1979) gave separate consideration to idiopathic and predisposed subjects. Predisposition was based on a history of thrombosis or embolism, a history of vascular, blood, or metabolic disease, or surgery or trauma. The relative risks from the four studies are in reasonable agreement ( p = 0.28),giving rise to a summary RR estimate of 5.8 [4.3,7.9] which is somewhat larger than that mentioned above from cohort studies.

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

327

Vessey et al. (1970) and Greene and Sartwell (1972) studied the relationship of OC use to postoperative venous thromboembolism, with other surgical patients as controls. Predisposition in the Stolley et al. (1975) study, as mentioned above, was not so specific, possibly explaining the somewhat, but not signficantly, lower RR estimate indicated in Table XII. At any rate, there is evidence of increased risk of nonfatal venous thromboembolism among current OC users for both predisposed and nonpredisposed women, with a relative risk that appears to be lower with predisposition and hence less than multiplicative. Part (b) of Table XI1 shows that rather similar RR estimates arise when the analysis is restricted to nonfatal pulmonary embolism. Specifically, a summary relative risk estimate of 5.0 [2.8,8.8] arises for idiopathic disease as compared to 2.7 [1.8,4.1] for predisposed disease from Maguire et al. (1979). Inman and Vessey (1968) studied fatal pulmonary embolism. An age- and parity-adjusted relative risk estimate of 8.3 [3.7,18.6] for current OC use arose among nonpredisposed subjects, with the definition of predisposition based on such conditions as prolonged immobility, recent surgery, hypertension, or diabetes. The corresponding relative risk estimate of 1.4 among predisposed subjects (excluding women who were pregnant or in the puerperium) is not significant. The final part (d) of Table XI1 concerns venous thromboembolism without pulmonary embolism. Current OC relative risk estimates of 4.7 for idiopathic disease and 1.5 for predisposed disease are obtained for current OC use, in good agreement with those for pulmonary embolism. Maguire e t al. (1979) give separate RR estimates for pulmonary embolism only, for venous thrombosis only, and for venous thrombosis and pulmonary embolism combined. There is little evidence of difference among the three RR estimates for idiopathic disease ( p = 0.82) or for predisposed disease ( p = 0.11). Throughout the literature on venous thromboembolism, concern has been expressed relative to a possible higher diagnosis rate among OC users compared to nonusers. The similar RR estimates for fatal compared to nonfatal pulmonary embolism argue against any important diagnostic bias. A formal test of equality of the nonfatal (5.0) and fatal (8.3)RR estimates for idiopathic pulmonary embolism is not significant ( p = 0.53). The fatal and nonfatal data combine to give an RR estimate and 95% CI of 5.9 [3.7,9.41. Similarly the two RR estimates are not significantly different among predisposed subjects and combine to give a summary RR estimate and confidence interval of 2.3 [ 1.6,3.3]. A number of reports have given venous thromboembolism rates in

TABLE XI1 OWL CONTRACEPTIVES AND PEHIPHERAL VASCXJLAH DISEASE: CASE-CONTROL STUDIES -

~~

CaSeS/UJIltrOlS tQ W QI

Source

(a) Nonfatal venous thromboembolism Idiopathic: Vessey and Doll (1969) Sartwell et al. (1969) BCDSPc (1973) Stolley et al. (1975), Maguire et al. (1979) Predisposed: Vessey et al. (1970) Creene and Sartwell (1972) Stolley et al. (1975), Maguire et al. (1979)

Current users

Neverlformer users

~-

RR estimate (95% CI),n current users

Combined RR estimate (95% CI), current users

42142 23/45 59/21 971135 31/170 121672 (89 cases, 632 controls)

6.3 [3.4,11.6] 4.Zb [2.2,17.8] 11.0 [5.2,25.0] 5.2d[3.2,8.61

1219 18/51 21/16 39/80 (318 cases, 670 controls)

3.8 [1.4,10.5] 6.5h[1.5,28.81 2.1d [1.5,3.0]

2.3 [1.7,3.2] ( p = 0.21)

2119 16/32 (44 cases, 632 controls)

4.0b [1.4,11.1] 5.5d [2.7,10.9]

5.0 [2.8,8.8] ( p = 0.61)

5.8 [4.3,7.9] ( p = 0.28)

(b) Nonfatal pulmonary embolism Idiopathic:

Sartwell et al. (1969) Stolley et al. (1975), Maguire et aZ. (1979)

Predisposed: Stolley et al. (1975), Maguire et al. (1979) (c) Fatal pulmonary embolism Idiopathic: Inman and Vessey (1968) Predisposed: Inman and Vessey (1968)

(168 cases, 670 controls)

16/171 9/171

101827 401827

(d) Other nonfatal venous thromboembolism (without pulmonary embolism) 38112 811107 Idiopathic: Sartwell et al. (1969) Stolley et al. (1975), (45cases, 632 controls) Maguire et al. (1979) Predisposed: Stolley et al. (1975), (150 cases, 670 controls) Maguire et al. (1979) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

2.7d [1.8,4.1]

8.3 [3.7,18.6] 1.4 [0.7,2.9]

4.3b[2.0,9.2] 5.0 (2.4,10.3)

4.7 [2.7,7.9] ( p = 0.79)

1.5 (0.9,2.6) ~~~~~

[ 1 Implies confidence interval calculated using approximate methods described in Section 11. RR estimate and confidence interval based on matched pair analysis. Boston Collaborative Drug Surveillance Program. d Log-relative risk estimate obtained as variance weighted average of log-relative risks for diagnostic subcategories.

330

ROSS L. PRENTICE AND I>AVIU B. TIIOhlAS

more detail by anatomic site. For example, RCGP (1978) provide current OC use relative risks of 4.2 (2.1,10.9), 2.4 (1.4,2.7), and 2.9 [0.9,9.0] for deep vein thrombosis of leg, superficial vein thrombosis of leg, and thrombosis of other (and unspecified) sites, respectively. The fact that these are not significantly different ( p = 0.47), and the fact that the largest RR estimate arises in a more serious site suggest further that bias is an unlikely explanation for the elevated risk among OC users (RCGP, 1978). In summary then, idiopathic venous thromboembolism arises with about a 5-fold increase among current OC users. Predisposed venous thromboembolism appears to be associated with a lesser relative risk, perhaps in the vicinity of 2.5, depending on the definition of predisposition. The studies summarized above do not provide evidence for an altered venous thromboembolism risk among former OC users. The reader is referred to RCGP (1967), Ludwig (1970), Fuertes de la Haba et al. (1970,1971), Grounds (1974), Sagar et aE. (1976), Hoover et al. (1978), Diddle et al. (1978), and Porter et aE. (1982) for additional information on OCs and venous thromboembolism. Some of these papers include additional cohort or case-control data. such data have not been included in our tables because of small numbers of disease events or the inability to classify disease events into the desired categories (e.g., to separate venous thromboembolism from cerebral or coronary thrombosis, or to separate predisposed from idiopathic disease occurrence). Note, however, that RCGP (1967) reported on a case-control study while all other RCGP reports cited in this article are based on the cohort previously described. The case-control study was the first to focus attention on venous thromboembolism as a possible consequence of OC use. Also, an updated report on venous thromboembolism in the Oxford cohort study (Vessey et al., 1986a) appeared as this article was nearing completion.

2. Dependence of Peripheral Vascular Disease Relative Risks on OC Dose and Formulation, Duration of Use, Time since Cessation of Use, and on Other Factors The presentation here will be somewhat briefer than that given for cerebral and coronary disease, since the dependence of peripheral vascular disease relative risks on other factors has generally received rather limited attention. RCGP (1983) presents relative risks for peripheral arterial disease (exclusive of chilblains) as a function of duration of current OC use. The standardized disease incidence rate during the first 2 years of usage is 1.71 based on 41 disease events, while

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

331

if duration categories greater than 2 years are combined one obtains a

standardized disease rate of 1.12 based on 77 disease events. The ratio of these two along with approximate 95% CI is 1.5 [1.0,2.2], suggesting a relative risk that is somewhat higher during the first 2 years of use than subsequently. Note, however, that the division point of 2 years was chosen subjectively, so the issue of a decreasing relative risk with increasing duration of use in unresolved. Sartwell et at. (1969) present data that could be interpreted as suggesting a higher relative risk for idiopathic venous thromboembolic disease during the first few months of OC use than subsequently, though such a trend is not suggested by the Boston Collaborative Drug Surveillance Program (1973) report. In summary, the studies described above do not appear to suggest any important dependence of venous thromboembolism relative risk on duration of OC use. Kay (1982) considered the importance of progestogen dose in relation to peripheral arterial disease in the RCGP cohort. Among current users of formulations with 50 pg of ethinyl estradiol along with norethindrone acetate (NEA) the standardized rates are slightly higher at the higher NEA doses, but the number of events are small and the trend is far from significant ( p = 0.42).Among current users of formulations with 30 p g of ethinyl estradiol along with levonorgestrel all seven peripheral arterial events occurred among users of the higher dose progestogen (0.25 mg). The dependence of peripheral arterial disease relative risks on such factors as cigarette smoking habits and age does not seem to have been specifically reported. RCGP (1978) presented data on deep vein and superficial vein leg thrombosis among current OC users with 50 pg ethinyl estradiol along with NEA. These combine to give disease rates of 1.41 based on 9 events, 3.00 based on 23 events, and 2.25 based on 3 events, at NEA doses of 1,3, and 4 mg, respectively. Clearly no trend is evident with these small numbers. Meade et al. (1980) investigated this same relationship using a much larger number of disease events (139 superficial venous thrombosis, 722 deep vein thrombosis, 308 pulmonary embolism, 67 venous thromboembolism deaths) reported to the British Committee on Safety of Drugs. No association with NEA dose at any of these four disease categories could be detected, except for pulmonary embolism where the number of events at the highest NEA dose was noticeably smaller than expected. Similarly Meade et al. (1980) were unable to detect any difference between the reported rates of superficial venous thrombosis (11 events), deep vein thrombosis (137 events), or pulmonary embolism (62 events) between users of either 0.25 or 0.15 mg levonorgestrel preparations along with 30 pg ethinyl estradiol.

332

ROSS L. PRENTICE AND DAVID 3. THOMAS

If the progestogen potency is assumed to be unimportant with respect to venous thromboembolism then varying estrogen doses may be directly related to OC relative risks. RCGP (1983) compared leg vein thrombosis rates among preparations with 50 pg or less, 75 or 80 pg, and 100 or 150 pg of estrogen. Upon combining deep and superficial vein thrombosis the trend toward higher incidence with greater estrogen dose is suggestive but by no means definitive ( p = 0.09).No difference between formulations with 30 and 50 p g of estrogen is suggested in Meade et al. (1980) for the incidence of superficial vein thrombosis, deep vein thrombosis, or pulmonary embolism. There is, however, a suggestion ( p = 0.06) that deaths from venous thromboembolism occur at a higher rate with the 50 pg dose. Inman et al. (1970), using reports to drug safety committees in the United Kingdom, Sweden, and Denmark, observed a significant trend in the reported rates of both fatal and nonfatal pulmonary embolism with increasing dose (50,75-80, 100, and 150 pg of the estrogen mestranol. However, see Kierkegaard { 1985)for a discussion of the limitations of such data on deep vein thrombosis. The epidemiologic data do not allow one to disentangle estrogen and progestogen roles in venous thromboembolism occurrence in a satisfactory manner. It seems reasonable to assume, however, that at least the estrogen potency, if not the potencies of both the estrogen and the progestogen, is important. As with the other cardiovascular diseases reviewed above, the venous thromboembolism relative risk does not appear to depend much on age or cigarette smoking. Maguire et al. (1979), for example, display relative risk estimates that are very similar among women less than 35 years of age as compared to older women, for both idiopathic and predisposed venous thrombosis. In the same analysis, relative risk estimates were somewhat, but not significantly, lower among smokers than among nonsmokers, for both idiopathic and predisposed disease occurrence.

D. CIRCULATORY SYSTEM DISEASES: POSSIBLE MECHANISMS A substantial literature exists on the relationship between OC use and a range of physiologic and pathologic processes that are relevent to cardiovascular disease. We will not attempt a comprehensive review of such literature here. Instead OC effects on certain major cardiovascular disease risk factors will be highlighted, and some comments will be given on the extent to which these effects might “explain” the relative risk patterns described earlier in this section.

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

333

OC use, as well as use of menopausal steroid preparations, has substantial influences on lipid and lipoprotein metabolism. For example, Bradley et al. (1978) analyzed serum levels of high-density-lipoprotein (HDL) cholesterol, which has been shown to be negatively related to ischemic heart disease occurrence, in a series of 4,978 women in the Walnut Creek cohort. HDL cholesterol levels were found to relate negatively to progestogen dosage at fixed estrogen dosage and to relate positively to estrogen dosage. Significant reductions to HDL cholesterol, in the vicinity of 10-20%, were observed for a number of combined OC preparations, particularly those with high progestogen content. Hennekens et al. (1979) observed an approximate 30% increase in fasting triglyceride concentrations among current OC users compared to nonusers. They also found that OC-induced lipid changes were reversible. Wahl et al. (1983) found that women taking OCs having a relatively low dose of estrogen along with a relatively high dose of progestogen had a 24% higher median concentration of low-density-lipoprotein (LDL) cholesterol, along with a significantly lower concentration of HDL cholesterol, than did nonusers. As in Bradley et al. (1978), women using OCs that have high dosages of estrogen and low dosages of progestogen had elevated HDL levels. Furthermore postmenopausal women taking estrogens were found to have 10-20% higher HDL cholesterol levels (Bradley et al., 1978) and 19% lower LDL cholesterol levels (Wahl et al., 1983), as compared to postmenopausal women not taking estrogens. These observations further implicate the progestational component of combined OCs with respect to ischemic heart disease occurrence. In terms of effects on lipid and lipoprotein cholesterol concentrations these reports suggest a possible benefit corresponding to the estrogen component of the OC. In fact, there is now accumulating evidence that postmenopausal estrogen use confers a moderate degree of protection from coronary artery disease (e.g., Henderson et al., 1986). A number of studies have reported somewhat higher levels of both systolic and diastolic blood pressure among OC users as compared to nonusers. For example, Cook et al. (1985) in a longitudinal study observed an average 4.1 mm Hg increase in systolic pressure among women starting OC use which was not significantly effected by duration of use. A similar average reduction (4.4 mm Hg) followed cessation of OC use. Women starting OC use were found to experience a reversible diastolic blood pressure increase of about 0.5 mm Hg per year of OC use. Similarly Fisch and Frank (1977) found a 5-6 mm Hg average elevation in systolic and a 1-2 mm Hg average elevation in

334

HOSS L. PRENTICE AND DAVID H . THOMAS

diastolic blood pressure in a study of the Walnut Creek cohort. By comparing blood pressure elevations among users of OC preparations having an identical 30 p g ethinyl estradiol component but with varying dosages of the progestogen levonorgestrel, Khaw and Peart (1982) provided data indicating the importance of the progestogen component of OCs in increasing blood pressure. Oral contraceptives have also been found to affect other systems of importance for cardiovascular disease. Specifically OC use has been reported to bring about modest decrease in glucose tolerance, a known risk factor for ischemic heart disease. Finally, and particularly relevant to thromboembolic disease, OC use has been found to bring about a reversible increase in the coagulability of blood, involving changes in the platelet, coagulation, and fibrolytic systems and changes in the structure of veins and arteries. Research on these topics is reviewed in some detail in Stadel (1981a,b). The modest average blood pressure elevations mentioned above at first sight do not seem able to explain much of the above-noted approximate 2-fold increase in subarachnoid hemorrhage incidence among current or former OC users, the approximate 5- and 2-fold respective increases in thrombotic stroke incidence among current and former OC users, or the approximate 2-fold and small respective increases in myocardial infarction incidence among current and former OC users. Three points, however, need to be kept in mind: First the relative risks for stroke and coronary heart disease appear to depend more strongly on blood pressure levels at younger ages (e.g., <SO years) than at older ages (e.g., Prentice et al., 1982),and the data at older ages tends to dominate most published reports on these relationships. Second, OC-induced blood pressure elevations are undoubtedly heterogeneous across individuals, giving rise to possible greater consequences for disease risk than would common blood pressure shifts. Third, and most important, blood pressure readings possess substantial measurenient error so that relative risk functions that relate disease occurrence to measured blood pressure levels may be very much flatter than are those that would arise if some “true” blood pressure level were substituted. In a forthcoming paper, Prentice (1987) brings together published data and plausible assumptions in respect to these points and illustrates that blood pressure effects alone may well be able to explain much, if not all, of the hemorrhagic stroke effects among current and former OC users and also the thrombotic stroke effects among former OC users. Additional factors are evidently important to explain the highly elevated thrombotic stroke risk among current OC users. Intui-

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

335

tively OC effects of blood coagulability seem an important aspect here, and they also seem important to an explanation of the elevated incidence of venous thromboembolism among current OC users. Blood pressure effects appear able to explain only a minority of the increased myocardial infarction incidence among current OC users, while blood pressure effects, or other changes such as blood lipid or lipoprotein changes that are also thought to promote atherogenesis, provide an explanation for any elevated myocardial infarction risk among former OC users. Effects on blood lipids and lipoprotein concentrations are, of course, major risk factors for coronary heart disease (but not for stroke), and the OC effects on these concentrations and, particularly, OC effects on blood coagulability seem a plausible explanation for much of the myocardial infarction elevation among current OC users. V. Oral Contraceptives and Cancer

A. ENDOMETRIAL CANCER It has been well documented that exogenous estrogens induce endometrial hyperplasia, including (precancerous) adenomatous hyperplasia, and strongly enhance risk of endometrial cancer (Weiss, 1983). The increase in risk of endometrial cancer has been observed in women treated at menopause with conjugated estrogens, ethinyl estradiol, and diethylstilbestrol (DES) and in women who received estrogen therapy for breast cancer and gonadal agenesis. There is strong evidence that sequential oral contraceptives, which exert a net estrogenic effect on the endometrium, also increase the risk of endometrial cancer. There are reports of adenomatous hyperplasia developing in women on sequential OCs (Lyon and Frisch, 1976; Kreutner et al., 1976), and the severity of the lesions have been related to duration of use. There have also been case reports of carcinoma in situ and invasive adenocarcinoma of the endometrium in users of sequential OCs (Lyon, 1975; Cohen and Deppe, 1977; Kelley et al., 1976). More importantly, three case-control studies (Table XIII) have provided evidence for an increased risk of endometrial cancer in users of sequential OCs (Weiss and Sayvetz, 1980; CASH, 1983a; Henderson et al., 1983a). The results of these studies are remarkably similar and together indicate that risk of endometrial cancer is approximately doubled in women who have ever used sequential OCs. One study (Weiss and Sayvetz, 1980) showed that the risk was confined to

336

ROSS L. PRENTICE AND DAVID B. THOMAS

TABLE XI11 RELATIVERISKSOF ENDOMETRIAL CANCER I N WOMEN Wno EVER USED SEQUENTIAL ORALC O N T R A C E ~ ICASE-CONTROL VES: STUDIES Cases/controls Source

Users

Nonusers

Weiss and Sayvetz (1980) CASH (1983a) Henderson et id.(1983a) Summary R R

7/19 5/26 1116

110/376 991540 1161121

-

-

RR estimate (95% CI)" 2.2 (0.6,7.3) 2.1 (0.8,5.8) 1.9 [0.7,5.3] 2.0 [1.1,3.8Ib

1 Indicates confidence intervals calculated using the approximate methods described in text. p Value of x 2 test for heterogeneity equal to 0.98. users of Oracon, a product that consisted of a particularly strong estrogen and a weak progestogen, but this was not noted in two other investigations (Henderson et al., 1983a; Kelsey et al., 1982). These discrepancies may be d u e to chance as a result of small numbers of Oracon users in all three studies. The increase in risk of endometrial cancer in users of sequential OCs has some relevance for clinicians who wish to treat women for menopausal symptoms with estrogens cycled with monthly administration of a progestogen. These findings suggest that such administration of a progestogen might not be sufficient to eliminate the increased risk of endometrial cancer associated with estrogen use. Some studies do suggest that progestogens reduce the risk of endometrial adenomatous hyperplasia and carcinoma in estrogen users (Gambrel1 et al., 1979; Greenblatt et al., 1982), although others do not (Schiff et al., 1982).Some of these investigations are not of rigorous design, and the dose and duration of use of progestogen given cyclically that is needed to eliminate completely the excess risk in estrogen users needs more careful quantitative evaluation. Estrogens probably act as promoters in a late stage of endometrial carcinogenesis by stimulating cell division. Progestogens reduce DNA synthesis in the endometrium (Lane et a!., 1983) and when given alone tend to lead to endometrial atrophy (Gopalkrishnan and Virkar, 1980). Therefore, giving enough progestogen for a sufficient period of time with an estrogen should reduce the risk of endometrial cancer. This is what has been found in studies of combined oral contrace pt ives . Table XIV shows results from five case-control studies, all but the most recent conducted in the United States. Together, they show a

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

337

TABLE XIV RELATIVERISKSOF ENDOMETRIAL CANCER IN WOMEN WHOEVER USED COMBINED ORALCONTRACEPTIVES: CASE-CONTROL STUDIES Caseslcontrols Source

Users

Nonusers

RR estimate (95%CI)“

Weiss and Sayvetz (1980) Kaufman et al. (1980) Hulka et al. (1982) CASH (1983a) La Vecchia et al. (1986) Summary RR

17/76 16/99 5/31 321525 71178

93/173 136/411 741172b 991540 16311104 -

0.5 (0.1,l.O) 0.4 (0.2,0.8) 0.4 [0.2,1.2] 0.5 (0.3,0.8) 0.6 (0.2,1.3) 0.5 [0.3,0.71”

-

[ 3 Indicates confidence intervals calculated using the approximate methods described in the text. b Includes women who used OCs for less than 6 months. p Value of ,y2 test for heterogeneity equal to 0.95.

reduction in risk of endometrial cancer of approximately 50% in women who have ever used combined oral contraceptives. Five studies with sufficient data also showed a decrease in risk with the duration of use (Table XV). The suggested heterogeneity among relative risks in the longest use category in Table XV presumably is explained by the differing durations of “longest” use among studies. Hulka et al. (1982) found the greatest reduction in risk to be in users of pills with the highest progestogen content, but this was not observed by Henderson et al. (1983a). Only the CASH study (1983a)provided information on risk after cessation of use. The RR after 10 or more years since exposure was estimated to be 0.8 (0.5, 1.3).

B. BREASTCANCER The possible influence of combined oral contraceptives on risk of breast cancer has been studied intensively during the past two decades. Table XVI shows results from 16 case-control studies of this topic. None show a significant alteration in risk in women who had ever used OCs, and the combined estimate of the RR is 1.0. Table XVII shows results from four cohort studies, two in Britain and two in the United States. None of these studies revealed a significantly altered risk of breast cancer for all ages combined, and the summary RE based on all four studies was again 1.0, identical to that based on the case-control studies (Table XVI). The RCGP study (1981b) showed an increased relative risk in women who were under 35 years of age. This

338

ROSS L. PKENTICE AND DAVID B. THOMAS

TABLE XV RELATIVERISKSOF ENDOMETRIAL CANCER IN RELATIONTO DURATION OF USEOF COMBINED ORALCONTRACEPTIVES: CASE-CONTROL STUDIES

Source

Kaufman et al. (1980)

Hulka et al. (1982)

Kelsey et al. (1982)

Henderson et al. (1983a)

CASH (1983a)

Sunimary RR

Years of use 0 <1 1-2 23 0 <5 25 0 52.5 >2.5 0 <2 1-3 4-5 26 0 <1 1-5 >5 Longestb

Number of Subjects, caseslcontrols

1361411 5/14 6/32 5153 74/17?, 3/14 2/17 311256 4/42 2/44 67150 23/22 12/11 419 4/18 991540 33J175 161266 11/251 -

p Value

RR estimate (95%CI)" 1.o 0.8 [0.3,2.3] 0.5 [0.2,1.2] 0.3 [0.1,0.8] 1.0 0.6 [0.2,2.2] 0.3 [0.1,1.3] 1.0 0.9 [0.3,2.7] 0.5 [0.1,2.2] 1.0 0.7 [0.4,1.4] 0.8 [0.3,2.0] 0.3 [0.1,1.0] 0.1 [0.1,0.3] 1.0 1.1 (0.7,1.8) 0.4 (0.2,0.7) 0.6 (0.4,O.g) 0.5 [0.3,0.6]'

of x 2 test for trend 0.05

>0.05

0.17 .CO.OOl

0.002

[ 1 Indicates confidence intervals calculated using the approximate methods described in the text. Longest use category in each study. p Value of x2 test for heterogeneity equal to 0.05.

finding, however, was not confirmed by Vessey et al. (1981b). Rates of breast cancer in nonusers who were less than 35 years of age in the RCGP study were unusually low, and this may be responsible for the observed increased relative risk in that study. In addition, the increased relative risk was only of borderline statistical significance. On balance, there does not appear to be an overall increase in risk of breast cancer in women who have ever used oral contraceptives. Table XVIII summarizes information from 13 case-control studies on long-term use of OCs. No consistent alteration in risk of breast cancer in long-term users is evident. However, the discrepant results among studies, and the significant p value for the x2 test for heterogeneity, suggest the need for filrther evaluation of risk in women with prolonged use. Table XIX summarizes results from eight case-control studies that

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

TABLE XVI RELATIVE RISKSOF BREASTCANCER IN WOMEN WHOEVER USED ORAL CONTRACEPTIVES : CASE-CONTROL STUDIES Caseslcontrols Source

Users

Nonusers

Henderson et 01. (1974) Paffenbarger et al. (1977) Sartwell et al. (1977) Kelsey et al. (1978) Ravnihar et al. (1979) Pike et al. (1981) Kelsey et al. (1981) Brinton et al. (1982) Harris et al. (1982) Vessey et al. (1983a) Hennekens et al. (1984) Rosenberg et al. (1984) Talamini et al. (1985) CASH (1986) Paul et al. (1986) LaVecchia et al. (1986) Summary RR

59169 2261398 22134 40134 30165 1351218 301141 2241189 361189 5371554 27312641 39712558 15/23 274312802 3101708 1041178

2481238 2261474 2621333 59165 1601315 28152 30011207 7381669 731279 6391622 71617260 79412468 3531351 187011774 1231189 67211104

-

-

RR estimate (95% CI)" 0.7 [0.5,1.2] 1.1 [0.9,1.4] 0.9 (0.5,1.5) 1.6 (0.8,2.4) 0.9 [0.6,1.5] 1.2 [0.7,1.9] 0.9 (0.6,1.3) 1.1 (0.8,1.4) 1.0 (0.6,1.4) 1.0 (0.8,1.2) 1.0 (0.9,1.2) 0.9 (0.8,l.l) 0.7 (0.4,1.4) 1.0 (0.9,l.l) 0.9 (0.9,1.25) 1.1 (0.8,1.5) 1.0 [0.9,1.1]*

[ ] Indicates confidence intervals estimated from published data. p Value of x2 test for heterogeneity equal to 0.73.

TABLE XVII CANCER IN WOMEN WHO EVER USED ORAL RELATIVE RISKSO F BREAST CONTRACEPTIVES: COHORTSTUDIES Cases per 1000 person-years (number of cases) Source

Ever-users

Vessey et al. (1981b) Trapido (1981) RCGP (1981b) Lipnick et al. (1986) Summary RR

0.50 (39) 0.93 (85) 0.47 (75) 1.39" (214) -

Never-users

0.52 (33) 1.11 (370) 0.39 (58) 1.34b(356)

-

RE estimate (95% CI)" 1.0 (0.6,1.6) 0.8 (0.7,1.2)

1.2 (0.8,1.7) 1.0 (0.8,1.3) 1.0 [0.8,1.1Ic

a [ 3 Indicates confidence intervals calculated using the approximate methods described in the text. b Age-adjusted incidence rates assuming a 4-year average follow-up. c p Value of x2 tests for heterogeneity equal to 0.51.

339

Caseslcontrols Source Ravnihar et al. (1979) ImVecchia et aI. (1986) Pike et al. (1981) Kelsey et ul. (1978) Harris et aZ. (1982) Paffenbarger et al. (1977) Vessey et aZ. (1983a) Brinton et al. (1982) Hennekens et ul. (1984) Rosenberg et al. (1984) Paul et al. (1986) Meirik et al. (1986) CASH (1986) Summary RR ~

Minimum years of use

Age of cases

Long-term users

2

20-49

2

<60 532

14129 41169 60177

4 5

5 8 8

10 10

10 10 12

15

-

20-44 35-54 <50 16-50 Premenopause

30-55 20-59

25-54 <50 20-54

~~

0

[ ] Indicates confidence intervals estimated from published data. p Value of x2 test for heterogeneity equal to 0.02.

916

17/99 17/26 66166

26121 121167

251128 631130 39/23 45/69

Nonusers

160/315 67211104 28/52 59/64 731279 104l195 6391622 2841237 71617260 79412468 1231189 961156 187011774

-

R R estimate (95% CI)" 0.9 [0.5,1.3]

1.1 (0.7,1.6) 1.6[0.9,2.8] 1.7(0.5,5.4) 0.8(0.5,1.4) 1.7[0.9,3.3] 1.0(0.7,l.S) 1.1 [0.6,1.91 0.7(0.4,1.3) 0.8(0.5J.3) 1.0 [0.7,1.5] 2.2(1.2,4.0) 0.6(0.4,O.g) 1.0[0.8,1.11"

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

341

TABLE XIX RELATIVE RISKSOF BREASTCANCER LONGAFTER INITIALUSE OF ORALCONTRACEWIVES: CASE-CONTROL STUDIES Caseslcontrols Source

Minimum years since first use

Past users

Nonusers

10 12 13 15 15 15 20 20

63172 1121154 56/63 15145 1011347 2171431 Not given 41/34

67211104 639/622 7381669 731279 79412468 1231189 1870/1774 961156

RR estimate (95% CI)" _ _ _ _

LaVecchia et al. (1986) Vessey et al. (1983a) Brinton et al. (1982) Harris et al. (1982) Rosenberg et al. (1984) Paul et al. (1986) CASH (1986) Meirik et al. (1986) Summary RR

-

_

a

_

_

1.4 (1.0,2.1) 0.7 (0.5,l.O) 0.8 [0.6,1.2] 1.4 (0.8,2.4) 1.1 (0.9,1.4) 0.9 [0.7,1.2] 0.8 (0.7,l.O) 1.1 (0.6,Z.O) 0.9 [0.8,1.0Ib

~

[ ] Indicates confidence intervals estimated from published data. p Value of x2 tests for heterogeneity equal to 0.03.

attempted to assess risk long after initial exposure to combined OCs. No consistent evidence for an increased risk after 10-20 years since first exposure is evident. The relative risks of 0.7 and 0.8 in the Vessey et al. (1983a) and CASH (1986) studies and of 1.4 in the LaVecchia et al. study (1986) were of borderline statistical significance, but no trends with time since initial exposure were found. Although the summary RR of 0.9 was also of borderline significance (95% CI from 0.8 to 1.0) so was the corresponding x2 test for heterogeneity, suggesting that caution should be exercised in interpreting this summary relative risk estimate. Results from two cohort studies (Table XX) do not show TABLE XX RELATIVE RISKSOF BREASTCANCER LONGAFTER INITIALUSE OF ORALCONTRACEPTIVES: COHORTSTUDIES Cases per 1000 person-years (number of cases) Source

Minimum years since first use

Trapido (1981) Vessey et al. (1981b) Summary RR

10 12 -

Past users

Nonusers

1.13 (31) 1.11(370) 0.82 (7) 0.52 (33)

-

-

RR estimate (95% CI)" 1.0 [0.7,1.5] 1.6 [0.7,3.6] 1.1 [0.8,1.5Ib

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. p Value of xe test for heterogeneity equal to 0.34.

342

ROSS L. PHENTICE AND DAVID B. THOMAS

a significant alteration in risk long after initial exposure to OCs. In the aggregate, the information summarized in Tables XIX and XX suggest that use of OCs does not influence risk of breast cancer after one to two decades from initial exposure. The question has been legitimately raised whether women at increased risk of breast cancer should take OCs. To answer this question, data have been analyzed to determine whether the relative risk of breast cancer is increased in OC users with specific known risk factors for breast cancer. Risk has not consistently been shown to be increased in users who have, for example, a history of benign breast disease, a family history of breast cancer, a late age at birth of first child, or no children. Risk in users has also not consistently been shown to vary among women of various ages. It is known that events early in a woman’s adult life alter her risk of breast cancer. Early menarche is associated with an increased risk, and risk is reduced by an early first pregnancy. Mechanisms for these associations are not clearly understood, but are presumably hormonal in nature. Therefore, it is reasonable to ask whether OCs used early in a woman’s life, or before her first pregnancy, might alter her risk of breast cancer. In 1981, Pike et al. reproduced an earlier finding by Paffenbarger et al. (1980) that, in women young enough to have used OCs before their first full term pregnancy, risk increased with duration of use before that event. Table XXI summarizes results from nine studies that addressed this issue. Three (Harris et ul., 1982; McPherson et al., 1983; Meirik et al., 1986) support the initial observations, and three do not (Vessey et al., 1982; Rosenberg et al., 1984; Paul et al., 1986). The CASH study (Stadel et al., 1985) yielded equivocal results, a small increase in risk in all duration categories of use, but no trend of increasing risk with months of exposure. Other studies, which provide only estimates of the relative risk in women who have ever used oral contraceptives before their first birth (Hennekens et al., 1984; Lipnick et ul., 1986),are not included in Table XXI. They found relative risks of 1.1 (0.8,l.ci) and 0.9 (0.6,1.2),respectively. The summary relative risk in Table XXI for long-term use prior to a first birth is 1.4 but, as expected, the p value of the x2 test for heterogeneity is small (0.002). In 1983 Pike et al. published an update of their earlier study. They did not present data on use before first full-term pregnancy, but claimed that use at an early age (regardless of whether before or after first pregnancy) was more important than use before a first-term pregnancy in increasing the risk of breast cancer, and they reported an increase in risk with duration of use before age 25. This observation

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

343

was not confirmed by a study in New Zealand (Paul et al., 1986) or the CASH study (P. Wingo, personal communication) but was supported by two studies in Sweden and Norway (Olsson et al., 1985; Meirik et al., 1986). No convincing explanation has been found for these inconsistent results regarding early use. A resolution of this issue is currently a topic of active investigation, and until these activities are successful, conclusions cannot be made regarding any influence on breast cancer risk of OC use prior to an initial full-term pregnancy or at an early age. Pike et al. (1983) also reported that risk was particularly increased in association with use before age 25 of OCs with a high progestogen potency as determined by the delay of menses test. However, this finding may be an artifact due to the erroneous classification of one particular product as being of high potency (Armstrong, 1986).Results from the CASH study (Stadel et al., 1985) were presented using the same classification and showed no evidence of an increase in risk in users of high progestogen potency preparations before the age of 25. Relative risks from that study associated with four categories of duration of use of high progestogen potency products, the longest being 273 months, varied from 1.1. to 1.3 but did not increase with length of exposure, and the 95% confidence limits of all estimates included unity. C. OVARIAN CANCER The findings from epidemiologic studies refer to all ovarian malignancies together, of which epithelial cancers predominate. Combined oral contraceptives have clearly been shown to reduce the risk of ovarian cancer. This is shown in Table XXII which summarizes the results from 10 case-control studies. The remarkable consistency of the findings among these studies suggests that combined OCs exert an actual protective effect against epithelial ovarian cancer. The summary relative risk of 0.6 suggests that risk is reduced by about 40% in women who have ever used OCs. The protective effect has been found to increase with duration of use (Table XXIII) but has been demonstrated even in short-term users. Based on results from the four studies shown in Table XXIII with users of 5 or more years’ duration, it is estimated that risk in women who have used OCs for more than from 5 to 9 years is approximately 40% that of nonusers. The protective effect appears to last for more than 10 years since last exposure, after which time risk has been shown to still be reduced by approximately 50% in two studies (Table XXIV). In 1982, Cramer et aE. pre-

TABLE XXI

RELATIVER ~ S K OF S BREASTCANCER IN

DURATION OF USE OF ORALCONTRACEPTIVES BEFORE Fmsr FULL-TERM PRECNANCY: CASE-CONTROL STUDIES RELATIONTO

Source

Age of cases at diagnosis

Months of use

Number of subjects, caseslcontrols

RR estimate (95% c1p

p Value of test for trend

Paffenbarger et 01. (1980)

Premenopausal

0 1-17 2 18 0 1-48 249 0 1-12 13-48 2-49 <12 13-48 249

57411146 1219 18/16 791141 531103 31126 9951996 28/25 18122 816 611249 4112 111 Not given

1.0 2.6 [1.1,5.91 2.6 [ 1.3,5.0] 1.0 1.0 [0.6,1.5] 2.5 (1.4,4.5) 1.0 0.8 [0.5,1.4] 0.7 [0.4,1.5] 0.9 [0.3,2.3] 1.o 3.8 (1.3,11.3) 12.9 (0.6,265.6) 1.0 1.2 (0.5,2.8) 1.7 (0.8,3.8) 3.1 (1.3,7.5)

Not given

Pike et u1. (1981)

S32

Vessey et ul (1982)

16-50

Harris et ul. (1982)

35-54

McPherson et u1. (1983)

545

0

1-12 13-48 249

0.009

0.01 <0.01

Rosenberg et al. (1984)

559

0 5 12

Stadel et al. (1985)

Paul et al. (1986)

Meirik et al. (1986)

Summary RE

544

25-54

<45

-

13-35 236 0 5 12 13-48 249 0 <24 25-47 48-71 2 72 0 536 37-95 296 Longest

64311946 141149 211133 10191 Not given

3251575 601147 26/82 11/41 11/52 2351338 1131119 45/52 29/18 -

1.0

0.8 (0.4,1.5) 1.3 (0.7,2.3) 0.9 (0.4,1.8) 1.0 1.3 (1.0,1.7) 1.1(0.9,1.5) 1.2 (0.9,1.6) 1.0 0.9 [0.6,1.3] 0.8 [0.5,1.3] 0.7 [0.4,1.4] 0.6 [0.3,1.1]

>0.05

>0.05

Not given

1.0

1.2 (0.8,1.7) 1.0 (0.6,1.7) 2.0 (1.0,4.2)b 1.4 [1.2,1.7]"

Not significant

[ ] Indicates confidence intervals estimated from published data. A lower 95% confidence interval of 1.8 was reported in the publication cited but was a typing error (0. Meirik, personal communication). p Value of x2 test for heterogeneity equal to 0.002.

346

ROSS L. PHENTICE AND DAVID B. THOMAS

TABLE XXII RELATIVERISKSOF OVARIAN CANCER IN "OMEN WHO EVER USED COMBINED ORALCONTRACEPTIVES: CASE-CONTROL STUDIES Caseslcontrois Source

Users

Nonusers

Newhouse et a/. (1977) Casagrande e t al. (1979) Willett et a/. (1981) Hildreth et al. (1981) Weiss et a/. (1981) Franceschi et UZ. (1982) Cramer et al. (1982) Rosenberg et al. (1982) CASH (1983b) LaVecchia et 01. (1986) Summary RR

19131 41I50 131152 '"3 211207 17/86 34148 331187 901683 391178

2811269 1091loo* 341312 591NG" 91/34Sd 1441475 110191 1031352 861921 3671 1104

-

-

RR estimate (95% CI)"

0.6 [0.3,1.1] 0.7[0.4,1.1] 0.8 (0.4,1.5) 0.5(0.2,1.7) 0.6 [0.4,1.0] 0.7(0.4,l.l) 0.4 (0.2,l.O) 0.6(0.4,O.g) 0.6(0.4,O.g) 0.6(0.4,l.O) 0.6[0.5,0.7le

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. Includes users of less than 6 month? duration. NG, Not given in published report. d Includes users of greater than 1 year duration. p Value of x2 test for heterogeneity equal to 0.96. 0

sented evidence that the protective effect was confined to women under 40 years of age. However, this finding was not confirmed by 1982; CASH, three other studies (Willett et al., 1981; Rosenberg et d.,

1983b). Women with infertility are at an increased risk of ovarian cancer and are less likely to be using oral contraceptives. This could lead to spurious inverse associations between OC use and ovarian cancer. This issue is examined in Table XXV. In all four studies summarized in this table, risk of ovarian cancer is decreased in users who are nulliparous. These associations, however, could overestimate the degree of protection because some nulliparous women are infertile and at reduced risk of ever having used OCs, and infertile women are at increased risk of ovarian cancer. However, the relative risks in OC users of proven fertility (i.e., parous women) are actually lower than in nulliparous users in two of the four studies. Also, the summary relative risk based on data from all four studies is nearly as low for parous as for nulliparous users; the values of these two estimates do not differ significantly ( p = 0.65 for x2 test for inequality of the two summary relative risks). In addition, the relative risks in the CASH (1983b) study were not appreciably altered after controlling directly

347

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

TABLE XXIII RELATIVE RISKSOF OVARIAN CANCER IN RELATION TO DURATION OF USE OF ORAL CONTRACEPTIVES: CASE-CONTROL STUDIES Source Casagrande et al. (1979) Willett et al. (1981) Weiss et al. (1981)

Rosenberg et al. (1982)

CASH (1983b)

Years of use

Number of subjects, caseslcontxols

<0.5 0.5-6 27

1091100 31136 10114 341311 7187 6165 911345 14179 3/83 4145 1031352 11/49 12/76 6151 861683 161106 131133 321213 101137 171332 36711104 291109 10169

0

<3 23 <1 1-3 4-8 29 0

<1 1-4 25 0

<0.25 0.25-1 1-2 3-4 25 LaVecchia et Summary RR

4Z. (1986)

0

52 >2 Longestb

-

RE estimate (95% CI)"

p Value of x 2

test for trend

1.0 0.7[0.4,1.2] Not given 0.6 [0.3,1.4] 1.0

0.7(0.3,1.7) 0.8 (0.3,2.1) 1.0 1.0(0.5,l.g) Not given 0.2 (0.1,0.6)

0.4(0.2,1.3) 1.0 0.9[0.5,1.8] 0.6[0.3,1.1] 0.3(0.1,0.8) 1.0 1.0(0.5,l.g) 0.7(0.4,1.4)

>0.05

0.002

0.8 (0.5,1.4) 0.5 (0.2,l.O) 0.4(0.2,0.6)

1.0 0.8(0.5,1.2) 0.5 (0.3,l.O) 0.4[0.3,0.6]"

0.02

a [ ] Indicates confidence intervals calculated using the approximate methods described in the text. b Longest use category in each study; Willet et al. and LaVecchia et al. studies omitted. c p Value of x2 test for heterogeneity equal to 0.74.

for history of infertility. It therefore is unlikely that the observed protective effect of OCs against ovarian cancer is spurious and due to incomplete control of the potentially confounding effect of infertility.

D. CERVICAL CANCER Cervical cancer is a particularly difficult disease to study with respect to oral contraceptives. It is generally accepted that invasive cervical cancer results from a series of changes in the cervical epithelium from normal epithelial structure to various grades of squamous dysplasia to carcinoma in situ and thence on to invasive cervical can-

348

ROSS L. PRENTICE AND DAVID B. THOMAS

TABLE XXIV RELATIVERISKSOF OVARIAN CAXCER BY TIME SINCE LASTUSEOF ORAL CONTRACEPTIVES: CASE-CONTROL STUDIES ~

Source

Years since last use

Number of subjects, caseslcontrols

Rosenberg et al. (1982)

Nonuser

CASH (l983b)

Nonuser

103/352 2/31 6/44 12/49 8/48 861683 15/54 151129 181221 261411 3671 1104 11/84 27/93 -

<1 1-4 5-9 5 10

<1 1-4 5-9 5 10 LaVecchia et al. (1986)

Summary RR”

Nonuser

<5 25 2 10

RR estimate (95% CI)”

1 .o 0.3 [0.1,1.3] 0.4 [0.2,1.0] 0.8[0.4,1.6] 0.5 [0.2,l.l] 1.0 1.0(0.4,2.2) 0.6(0.3,l.l) 0.5 (0.3,O.g) 0.5(0.3,O.g) 1.0 0.6(0.3,l.l) 0.8(0.5,1.2) 0.5 [0.3,0.81‘

0 [ 3 Indicates confidence intervals calculated using the approximate methods described in the text. Based on data from Rosenberg et al. and CASH studies only. c p Value of x 2 test for heterogeneity equal to 1.0.

cer. Oral contraceptives could act at any one stage to enhance progression to the next stage. Therefore studies need to be conducted that assess separately the influence of steroid contraceptives on risk of different stages of this condition. Many studies to date have included a mixture of lesion types. Furthermore, until recently there have been no good studies of invasive cervical cancer, and the relevance of findings from studies of preinvasive conditions to the assessment of the influence of OCs on risk of invasive cervical cancer is unclear. Another difficulty in assessing the influence of OCs on risk of cervical cancer is that cervical cancer is a disease that occurs particularly in women who have multiple sexual partners and perhaps also in women who have had early sexual experiences. In some cultures such women are more likely to use OCs than women with more conservative sexual behavior. Therefore, to assess accurately the influence of steroid contraceptives on risk of cervical cancer, studies must be conducted in which sexual behavior is carefully ascertained and taken into account in the data analyses. Even in studies where sexual behavior has been determined, the validity of such information is uncertain; if adequate sexual information is not obtained, then controlling for the confound-

TABLE XXV RELATIVERISKSOF OVARIAN CANCER IN PAROUS AND NULLIPAROUS WOMEN WHO HAVEEVER USED ORALCONTRACEPTIVES: CASE-CONTROL STUDIES Parous

Nulliparous

Caseslcontrols Source

Users

Nonusers

Willett et al. (1981) Cramer et al. (1982) Rosenberg et al. (1982) CASH (198313) Summary RR

121137 13/39 241159 721870

241270 76/76 761269 611618

-

-

Cases/controls

RR estimate (95% C1)a 1.0 (0.5,Z.O) 0.3 (0.2,0.7) 0.5 [0.3,0.9] 0.8 [0.4,1.3] 0.6 [0.5,0.8]

Users

Nonusers

1/15 2119 9/28 20/95

10/41 34/13 27/83 25/52

-

-

RR estimate (95% CI)” 0.3 (0.0,2.1) 0.9 (0.3,2.5) 0.6 [0.3,1.4] 0.3 (0.1,0.8) 0.5 [0.3,0.9]“

[ 1 Indicates confidence intervals calculated using the approximate methods described in the text. p Value of x2 test for heterogeneity equal to 0.13. p Value of x2 test for heterogeneity equal to 0.46.

350

HOSS L. PRENTICE AN11 DAVID €3. THOMAS

ing influence of this behavior will only partially correct estimates of relative risk in relation to OCs for the influence of sexual practices. A third problem in assessing the influence of steroid contraceptives on risk of cervical cancer is related to the influence of pap smears. If cases detected at screening are the ones that are more likely to be studied, and if women are more likely to have pap smears if they have used steroid contraceptives, then the cases that are studied may be more likely to have used OCs than other cases in the population. This could lead to spuriously elevated relative risks in relation to OC use in case-control studies. Conversely, if pap smears protect against invasive disease, then studies of these conditions could include relatively fewer users in the case group than in other cases in the population; and this could result in a spuriously low relative risk in relation to OCs. The influence of prior pap smears must therefore be considered in assessing risk of cervical cancer in relation to OC use. Finally, if users of OCs tend to have their pap smears interpreted by different individuals than nonusers, or if readers of pap smears have knowledge of a woman's use of OCs, then smears from users may tend to be diagnosed less (or more) conservatively than smears from non-users, and result in diagnoses of more (or fewer) advanced lesions in users. Consider first cervical squamous dysplasia. Results from four casecontrol studies are summarized in Table XXVI, and results from three cohort studies are shown in the middle portion of Table XXVII. An estimated relative risk greater than unity in women who ever used OCs was found in all seven studies, and values of the relative risks were not significantly heterogeneous. The two summary relative risks TABLE XXVI RELATIVERISKSOF

DYSPLASIA IN WOMEN WHO EVER U S E D COMBINED ORAL CONTRACEPTI\'ES: CERVICAL SQUAMOUS

CASE-CONTROL STUDIES Cases/controls Source

Users

Nonusers

RR estimate (95% CI)"

Thomas (1972) Ory et al. (1977) Fasal et al. (1981) Clarke et a!. (1985) Summary RR

42/103 68216363 521796 2361425 -

631199 17212190 4511639 14/75 -

1.2 [0.8,2.0] 1.4 [1.1,1.6] 1.7 [1.1,2.5] 1.7 [0.9,3.1] 1.4 [1.2,1.6Ib

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. p Value of x2 test for heterogeneity equal to 0.69.

TABLE XXVII RELATIVERISKSOF CERVICAL CARCINOMA in Situ AND SQUAMOUS DYSPLASIA I N WOMEN WHO EVER USED O R A L CONTRACEPTIVES: C O H O R T STUDIES

Cases per 1000 person-years (number of cases) Condition Carcinoma in situ

Squamous dysplasia

Both combined

a

Source

Users

Nonusers

RR estimate (95% C1)O

Summary RR (95% C1)CJ

Peritz et al. (1977) Vessey et a1. (1983b) Andolsek et al. (1983)

1.19 (29) 0.72 (47) 0.92 (45)

0.32 (6) 0.45 (12) 0.79 (52)

3.7 [1.5,9.0] 1.6 [0.8,3.0] 1.2 [0.8,1.7]

1.5 [1.1,2.0] ( p = 0.06)”

Peritz et al. (1977) Vessey et al. (198313) Andolsek et al. (1983)

1.19 (29) 0.77 (50) 0.88 (43)

0.24 ( 2 ) 0.53 (14) 0.78 (51)

5.0 [1.2,20.8] 1.5 [0.8,2.6] 1.1 [0.8,1.7]

1.3 [1.0,1.5] ( p = 0.13)b

Peritz et al. (1977) Vessey et al. (198313) Andolsek et al. (1983)

2.38 (58) 1.49 (97) 1.80 (88)

0.56 (8) 0.98 (26) 1.57 (103)

4.3 [2.0,8.9] 1.5 [1.0,2.3] 1.1[0.9,1.5]

1.4 [1.1,1.8] ( p = 0.004)”

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. p Value of x2 test for heterogeneity.

352

ROSS L. PRENTICE AND DAVID B. THOMAS

of 1.4 from the case-control studies and 1.3 from the cohort studies are remarkably similar. Table XXVIII shows that risk of squamous dysplasia was found to increase with duration of use in two ofthe three case-control studies in which years of exposure were considered. The summary relative risk estimate for dysplasia in users of the longest duration categories considered in each study was 1.9, but the p value of 0.006 for the x2 test for heterogeneity suggests that this summary value should be interpreted with caution. However, these results are supported by two cohort studies (the middle portion of Table XXIX)which show that the risk of squamous dysplasia in users of more than 4 years was found to be increased. Attempts to control for the potentially confounding effects of multiple sexual partners were made in the case-control studies of Thomas (1972) and Clarke et al. (1985), but not in those of Ory et aE. (1977)or Fasal et al. (1981),or in any of the three cohort studies. All these results, in spite of their relative consistency, could therefore be due to the confounding influence of sexual behavior on the relative risk estimates. Relative risks of carcinoma in situ in women who have ever used OCs, as estimated from five case-control studies, are shown in Table TABLE XXVIII RELATIVERISKSOF CERVICAL SQVAMOUS DYSPLASIA IN RELATIONTO DURATION OF USEOF ORALCONTRACEPTIVES: CASE-CONTROL S~UDIES Xumber of subjects, caseslcontrols

RR estimate

of use 0 1 2 3 24 0 52.5 >2.5 0 54 25 Longes tb

17212190 40814285 170/1368 801530 241180 4511639 231407 251373 14/75 101I228 1331192 -

1.0 1.2 [1.0,1.4] 1.6 [1.3,2.0] 1.9 [1.4,2.5] 1.6 [1.0,2.5] 1.o 1.7 [1.0,2.8] 1.6 [1.0,2.6] 1.0 2.4 [1.3,4.4] 3.7 [2.0,6.8] 1.9 [1.4,2.6]'

Years Source Ory et al. (1977)

Fasal et al. (1981)

Clarke et al. (1985) Summary RR

(95% CI)B

I I Indicates confidence intervals calculated using the approximate methods described in the text. Longest use category in each study. p Value of xg test for heterogeneity equal to 0.006.

TABLE XXIX RELATIVERISKSOF CERVICAL CARCINOMA in Situ AND SQUAMOUS DYSPLASIA IN WOMEN WHOUSEDORAL CONTRACEPTIVES FOR MORE THAN 4 YEARS: COHORT STUDIES Cases per 1000 person-years (number of cases) Condition Carcinoma in situ Squamous dysplasia

Both combined

(I

b

Source

Users

Peritz et al. (1977) Vessey et al. (1983b) Peritz et al. (1977) Vessey et al. (1983b) Peritz et al. (1977) Vessey et al. (1983b)

1.73 (17) 0.75 (32) 1.17 (12) 1.04 (47) 2.90 (28) 1.72 (79)

Nonusers

0.32 (6) 0.45 (12) 0.24 (2) 0.53 (14) 0.56 (8) 0.98 (26)

RR estimate (95% CI)"

Summary RR (95% CI)"

5.4 [2.1,13.7] 1.7 [0.9,3.2] 4.9 [1.1,21.8] 2.0 [1.1,3.6] 5.2 [2.4,11.4] 1.8 [1.1,2.7]

2.5 [ 1.4,4.3] (p = 0.05)b 2.2 [1.3,3.9] ( p = 0.2Qb 2.3 [1.6,3.4] ( p = 0.02p

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. p Value of x 2 test for heterogeneity.

354

ROSS L. PRENTICE AND DAVID B. THOMAS

TABLE XXX RELATIVERISKSOF CERVICAL C.~CINOM in A Situ IN WOMEN WHOEVEK USED COMBINED ORAL COhTRKEPTIVES: CASE-CONTROL STUDIES Caseslcontrols Source

Users

Nonusers

Thomas (1972) Worth and Boyes (1972) Boyce et al. (1977)b Ory et al. (1977) Fasal rt at. ( 1981)b Summary RR

301103 27 1 I570 3641371 12116363 231796 -

741199 39192 3251318 2612190 321 1639

-

RR estimate (95% CI)O 0.6 [0.4,0.9] 1.1 [0.8,1.7] 1.0 [0.8,1.21 1.6 [1.3,2.0] 1.1 [0.6,1.8] 1.0 [0.9,1.2]"

[ ] Indicates confidence i n t e n d s calculated using the approximate methods described in the text. b includes some invasive cervical cancers in case groups. p Value of x 2 test for heterogeneity equal to 0.04.

XXX. The results are inconsistent ( p value of x2 test for heterogeneity In only the study with the lowest relative risk estimate equal to 0.04). (Thomas, 1972) was the influence of multiple partners on risk considered, and differences in the degree of confounding for sexual behavior could be an explanation for this heterogeneity. The summary relative risk, although of questionable interpretation because of the variability in results among studies, is reassuringly equal to unity. However, results from three cohort studies, shown in the upper portion of Table XXVII, are not. All show relative risks of carcinoma in situ in women who ever used OCs to be greater than one, and the summary relative risk based on all three cohort studies is 1.5. Here again, however, the p value of the x2 test for heterogeneity is small (0.06), and sexual variables were not adequately considered in any of the three studies. Another study, conducted by Stern and Coffelt (1977), consisted of following 300 women with dysplasia forward in time. Two hundred three of these women had received OCs and 97 had not. Most of the latter had used an IUD. After 7 years of follow up, the probability of progression from dysplasia to carcinoma in situ was found by life table analysis to be 0.30 in the OC users and 0.05 in the nonusers, for a relative risk estimate of 6. In the analysis, the potential confounding influence of age at first intercourse, number of pregnancies, race, and education were controlled for, but no measures of numbers of sex partners were available. Relative risk estimates for carcinoma in situ in relation to duration of use, from three case-control studies, are presented in Table XXXI.

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

355

TABLE XXXI RELATIVE RISKSOF CERVICAL CARCINOMA in Situ IN RELATION TO DURATION OF USEOF ORAL CONTRACEPTIVES CASE-CONTROL STUDIES Years Source Ory et al. (1977)

Boyce et al. (1977)

Fasal et al. (1981)

Summary RR

of use

Number of subjects, caseslcontrols

0 1 2 3 =4 0 <2 2-4 25 0 52.5 >2.5

2512190 6014285 3511368 161530 101180 3251318 2041204 1021119 58/48 3211639 101407 121373

Longestb

-

RR estimate (95% CI)"

1.0 1.3 [0.8,2.1] 2.5 [ 1.5,4.2] 2.6 [1.4,4.9] 4.7[2.2,9.9] 1.0 1.0 [0.8,1.3] 0.8 [0.6,1.1] 1.2 [0.8,1.8] 1.0 1.0 [0.5,2.1] 1.2 [0.7,2.1] 1.5 [1.1,2.1Ic

[ 1 Indicates confidence intervals calculated using the approximate methods described in the text. * Longest use category in each study. p Value of x2 test for heterogeneity equal to 0.005.

Two of the three studies (Boyce et al., 1977; Fasal et al., 1981) show minimal increases in relative risk in users of more than 5 and 2.5 years' duration, and no graded increase with duration; the other (Ory et al., 1977) shows a strong increase in risk with duration of use. Although the summary estimate of relative risk in long-term users, based on data from the longest use category in each study, is 1.5 with confidence limits that do not include unity, the summary x2 for heterogeneity is highly significant, and the interpretation of this summary value is therefore unclear. Relative risks in users of more than 4 years' duration, as estimated from two cohort studies, are shown in the upper portion of Table XXIX. Both are greater than one, but the p value for the x2 test for heterogeneity is small, so that inconsistency of these results also makes their interpretation difficult. In none of the studies shown in Tables XXIX or X M I were adequate measures of sexual behavior considered as possible confounders. Four case-control studies of both dysplasia and carcinoma in situ combined revealed inconsistent estimates of relative risk in women who had ever used OCs (Table XXXII). The two studies that did not show an increase in risk (Thomas, 1972; LaVecchia et al., 1986) were the two in which an attempt was made to control for the confounding

356

ROSS L. PFiENTICE AND DAVID 8. THOMAS

TABLE XXXII RELATIVERISKSOF CERVICAL CARCINOMA in S i t U OR SQUAMOUS DYSPLASIA I N WOMENWHO EVER USEDCOMBINED ORALCONTRACEPTIVES: CASE-CONTROL STUDIES

Caseslcontrols Source

Users

Nonusers

RR estimate (95%CI)'

Thomas (1972) Ory et al. (1977) Fasal et al. (1981) LaVecchia et al. (1986) Summary RR

1171103 80216363 751796 71182

2091199 19812190 7711639 1311120 -

0.9 [0.7,1.3] 1.4 [1.2,1.6] 1.4 [1.0,1.9] 0.7 (0.4,l.l) 1.3 [1.1,1.4Ib

~

-

~~

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. b p Value of ' x test for heterogeneity equal to 0.01.

influence of sexual promiscuity. The summary relative risk of 1.3 should therefore be interpreted cautiously. The same is true for the relative risks estimated from the three cohort studies and the corresponding summary relative risks, all of which are greater than one. These are shown in the lower portions of Tables XXVII (for ever users) and M I X (for users of more than 4 years' duration). In summary, most studies of cervical intraepithelial neoplastic lesions and OCs have tended to show relative risks in ever-users and long-term users of OCs to be greater than unity. However, the results among studies have been inconsistent, with a few studies showing no alteration in risk, and the estimates of relative risks from the others varying significantly in magnitude. Most studies have not provided relative risk estimates that have been adequately controlled for the potentially confounding influence of sexual practices, and this may be one explanation for the reported increases in risks. Variations in the degree of confounding could also explain the inconsistencies in results. Even if oral contraceptives were found to increase the risk of dysplasia or carcinoma in situ, this would not necessarily mean that they also increase the risk of invasive cervical cancer. Many intraepithelial lesions do not progress to invasion. Results from studies of invasive cervical cancer must be assessed to answer directly whether OCs alter the risk of this condition. Table XXXIII summarizes results from three case-control studies. All three show an increase in risk in women who ever used OCs, and together they yielded a summary relative risk of 1.3. Detailed sexual histories were elicited in all studies, and the

357

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

TABLE XXXIII RELATIVE RISKSOF INVASIVE CERVICAL CANCER IN WOMEN WHOEVER USEDCOMBINED ORALCONTRACEPTIVES: CASE-CONTROL STUDIES Caseslcontrols Source

Users

Nonusers

WHO (1985) Brinton et al. (1986) LaVecchia et al. (1986) Summary RR

26511272 2691386 43132

44412663 2101403 1821193

-

RR estimate (95% CI)"

1.2 (1.0,1.4) 1.5(1.1,2.1) 1.7 (0.8,3.6) 1.3 [1.1,1.5Ib

-

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. p Value of x2 test for heterogeneity equal to 0.34. (I

relative risks shown were controlled for a variety of sexual variables and prior pap smears, as well as other potentially confounding factors. Results from three cohort studies are shown in Table XXXIV. In two of the studies (Vessey et al., 198313; Andolsek et al., 1983) invasive cervical cancer occurred only in us'ers of OCs and not in the comparison groups of nonusers, so relative risk estimates are indeterminant. In the third study (Kay, 1983), a doubling of risk was observed. Although in none of these studies was the potentially confounding influence of sexual behavior assessed, the consistency of the results, and the strength of the associations (although based on small numbers of cases) support the findings from the more rigorous case-control studies. Table XXXV shows that in all three case-control studies, risk increased with duration of use; and the relative risks in users in the longest use category in each study have confidence limits that do not TABLE XXXIV INVASIVE CERVICAL CANCERIN WOMENWHOHAVEEVER USEDORALCONTRACEPTIVES: COHORTSTUDIES Source

Exposure category

Ever OC Ever IUD Kay (1983) Ever OC Never OC Andolsek et al. (1983) Ever OC Never OC

Vessey et al. (198313)

Number of cases

Personyears

Cases per 1000 person-years

Estimated RR (95% CI)

13

65,101 26,432 203,613 146,534 48,687 65,637

0.20

Unbounded

0

34 10 6 0

0

0.15 0.07 0.12 0

2.1 (1.1,4.3) Unbounded

358

BOSS L. PRENTICE AND DAVID B. THOhlAS

TABLE XXXV RELATIVERISKSOF INVASIVE CERVICAL CANCER

IN

RELATIONTO DURATION

OF USE OF ORAL CONTRACEPTIVES: CASE-CONTROL STUDIES

Source

WHO (1985) Brinton et u l . (1986)

LaVecchia et al. (1986)

Summary RRb

Years of use

Number of subjects, caseslcontrols

RR estimate (95% a)"

0 1-5 >5 0

44412650 191/ 1024 631224 2691386 1141260 961143 182/193 28125 15/7

1 .0 1.1 [0.9,1.3] 1.5(1.1,2.1) 1 .0 1.3 (0.9,l.g) 1.9 [ 1.3,2.7] 1.0 1.3 (0.7,2.3) 2.9 (1.1,7.8)

1-5

>5 0 52 >2 0 1-5 >5

-

1.0

1.1 [1.0,1.3]" 1.7[1.3,2.1Id

a [ I Indicates confidence intervals calculated using the approximate methods described in the text. Based on data from WHO and Brinton et d.studies only. p Valiie of test for heterogeneity equal to 0.48. " p Value of xs test for heterogeneity equal to 0.38.

xz

include one. The estimates shown are controlled for the potentially confounding effects of several sexual variables and prior pap smears. Results from the cohort study of Vessey et al. (1983b) are compatible with these findings. In that study, rates of invasive cervical cancer did not increase until after 4 years of use of OCs and increased with duration of use thereafter. Additional studies are needed to confirm or refute these observations regarding OCs and invasive cervical carcinoma, and some are underway for this purpose. Smoking as a potential confounder, assessed in the studies by Brinton et al. (1986) and LaVecchia et a2. (1986)but not in the other investigations cited, needs to be considered in future endeavors. In addition, at least two ongoing studies have a serologic component. These will provide information on prior exposure of cases and controls to a variety of sexually transmitted diseases, such as herpes simplex virus type 11, cytomegalovirus, and possibly papilloma viruses. Antibodies against these viruses can be used as independent indicators of sexual behavior, and, if warranted, controlled for in the analyses to provide better estimates of relative risks in relation to use of OCs. Finally, if the husbands of users of OCs are more likely to have had extramarital sexual relations than the hus-

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

359

bands of nonusers, and if risk of cervical cancer is associated with male sexual behavior, then the observed observations of OCs to cervical cancer could be due to the confounding influence of male sexual behavior. Studies have been initiated to assess the role of the sexual practice of men in the genesis of cervical cancer. In summary, studies to date consistently show an increase in risk of invasive cervical cancer in users of oral contraceptives. These associations may represent a causal relationship, they may be due to incomplete control for the confounding influences of the sexual behavior of women or their male partners, or they may be due to other unrecognized sources of spurious associations. The evidence for the association being causal is not currently sufficient to warrant recommending that women should avoid OCs. Women at risk of sexually transmitted diseases who use OCs, like other women at risk of such conditions, should have periodic pap smears.

E. LIVERTUMORS Two histologic types of benign liver tumors have been associated with use of oral contraceptives, hepatic cell adenoma and focal nodular hyperplasia. The former consists of cells resembling hepatocytes but with vacuolated cytoplasm and little architectural organization. They are usually encapsulated and are highly vascular, with vessels distributed throughout the hepatocytes. The cells in lesions of focal nodular hyperplasia resemble more closely normal hepatocytes and show evidence of some organization. They are separated into lobules by fibrous septa and contain proliferating bile ducts. Blood vessels tend to be found within the septa. Some of these lesions have been called hamaratomas in some reports, but a slide review of a series of these lesions by a panel of pathologists, using standardized criteria, reclassified them as either hepatocellular adenomas or (more frequently) focal nodular hyperplasia and recommended that the term hamaratoma be reserved for mesenchymal hepatic lesions in children (Nime et al., 1979). A relationship between hepatic cell adenoma and use of oral contraceptives was first suggested by Baum et al. (1973), who reported a series of seven cases in women of childbearing age, all of whom had used OCs. Prior reports of this condition, before the OC era, were extremely rare. By contrast, there are a number of reports of focal nodular hyperplasia in both sexes before the advent of OCs. It is not known whether incidence rates of these latter tumors have increased in recent years because information on them is not usually included in tumor registries.

360

ROSS L. PRENTICE AND DAVID B. THOMAS

Subsequent to the report by Baum et al. (1973), a number of additional series of women with benign liver tumors have been published, which include individuals with both hepatic cell adenomas and focal nodular hyperplasia (Nissen et al., 1976; Christopherson and Mays, 1977; Warren and Bellward, 1979; Gonvers et al., 1978; Vana et al., 1979; Nime et al., 1979; Graf et al., 1980; Mahbouhi et al., 1981;Cavin et al., 1981; Barrows and Christoperson, 1983). A high proportion of the women in these series have been users of OCs, but the extent to which this is due to selective reporting of cases who were users is unknown; such reports are not strong evidence for a causal relationship between OCs and benign liver tumors. The largest of these series was assembled as a result of a national survey of primary liver tumors in 749 U.S. hospitals conducted in 1976 by the Cancer Commission of the American College of Surgeons (Vana et al., 1977; Nime et al., 1979). Observations from this series provide additional evidence that OCs may be a cause of hepatic cell adenomas. These lesions were found only in individuals without a known history of alcohol abuse or exposure to recognized hepatic toxins and arose in livers with no histologic evidence of hepatic cirrhosis or fibrosis. In addition, all occurred in women who had used OCs, and the size of the tumor was related to duration of use. These cases were largely women of reproductive age. In contrast, women with focal nodular hyperplasia in that series tended to be somewhat older than women with hepatic cell adenoma, and there were also some male cases. These lesions, too, however, occurred in the absence of hepatic cirrhosis or fibrosis. Also, the histologic features of the focal nodular hyperplasias in users and nonusers of OCs tended to differ. The lesions in users tended to have more peliosis (blood-filled spaces and cysts with complete or partial endothelial lining connected to liver sinusoids or veins), thrombosis, and scarring. The tumors in users were also larger than those in nonusers. It has been suggested that these tumors may arise as a result of the vascular changes induced by OCs, which result in ischemia and hepatic cell death followed by cellular regeneration and fibrosis which leads to lesions designated as focal nodular hyperplasia. Alternatively, OCs may merely induce vascular changes in preexisting tumors that lead to hemorrhage and symptoms, so that focal nodular hyperplasia is more likely to be diagnosed in users than nonusers. (Some liver cell adenomas also show evidence of peliosis, necrosis, hemorrhage, and thrombosis, which suggest that a similar selection bias may be operating, although this is less likely in view of the extreme rarity of these tumors in the absence of oral contraceptives.) Case reports of both

EPIDEMIOLOGY OF ORAL CONTRACEFTIVES

AND DISEASE

361

hepatic cell adenomas and focal nodular hyperplasias regressing in users of OCs after discontinuation of use, however, provide further evidence for a causal role of OCs in the etiology of both of these conditions (Kay, 1977; Mays et al., 1976; Klatskin, 1977; Emerson et al., 1980; Scott et al., 1984). No case-control studies of focal nodular hyperplasia have been conducted, but there have been two case-control studies of hepatic cell adenomas and OCs (Table XXXVI). Edmondson et al. (1976) interviewed 34 of 42 cases that occurred in the Los Angeles area and compared their responses with a control group that consisted of friends of these cases. They estimated a relative risk of 1.78 in relation to OC use, but this may be an underestimate because friends of cases might be more likely to use OCs than women in the general population since the cases were all OC users. More importantly, the cases tended to use OCs about twice as long as the controls, and the relative risk increased strongly with duration of use and was estimated to be 25.0 for users of more than 9 years’ duration (Table XXXVI). In the second study (Rooks et al., 1979), 79 women with liver cell adenomas were interviewed. Their reponses were compared to those of controls who were selected from women who lived in the same neighborhood as the cases. Three controls were selected for each case. As shown in Table XXXVI, the relative risk increased markedly with duration of use of OCs. Risk was found to be higher in women who were over 27 years of age than in younger women. Two cohort studies in Britain (Vessey et al., 1977a; RCGP, 1981b) TABLE XXXVI RELATIVERISKSOF LIVERCELLADENOMAS BY DURATION OF USEOF ORAL CONTRACEPTIVES: CASE-CONTROL STUDIES Source

Months of use

Number of subjects, casedcontrols

RR estimate

512 13-36 37-60 61-84 85-108 2 109 5 12 13-36 37-60 61-84 285

6/15 418 717 412 311 1011 71121 11/49 20123 21/20 2017

1.0 1.3 2.5 5.0 7.5 25.0 1 9 116 129 503

~

Edmondson et al. (1976)

Rooks et al. (1979)

362

ROSS L. PHENTICE AND DAVID €3. THOMAS

have not shown an increase in risk of either type of benign liver tumor, probably because the absolute risk is very low. A WHO expert committee (1978) estimated that the risk of these tumors is less than 3 per 100,000 OC users for women less than 30 years of age. It is higher for older women, but this risk has not been quantified. Risk has been estimated to be between 1 and 5 per 1,000,000 in nonusers. In summary, evidence is strong that hepatic cell adenomas are a rare complication of OC use. Evidence that this is true also for focal nodular hyperplasia is weak, and the reports of this lesion occurring in women using OCs could also be due either to selective diagnosis of these conditions in OC users as a result of steroid hormone-induced vascular changes and hemorrhage or to selective reporting of these tumors when they occur in OC users. Some lesions in women who have used OCs that have been diagnosed to be focal nodular hyperplasia (Davis et al., 1975; Terblanche, 1978) or hepatic cell adenoma (Goldfarb, 1976; Klatskin, 1977; Neuberger et al., 1980; Tesluk and Lawrie, 1981) have been reported to also have areas of hepatic cell carcinoma and evidence oftransition from the benign to malignant lesions. Also, a histologic variant of hepatic carcinoma, termed polygonal cell with fibrous stroma (Berman et al., 1980), has been described which may represent a transition lesion between focal nodular hyperplasia and well-differentiated hepatocellular carcinoma. These observations have raised concern that OCs may be causally related to primary liver cell carcinomas. There have been numerous reports of liver carcinomas occurring in users of OCs. These reports are primarily of ordinary hepatocellular carcinomas (Mays et al., 1976; Glassberg and Rosenbaum, 1976; Tigano et al., 1976; Pryor et a/., 1977; Schmidt, 1977; Menzies-Cow, 1978; Gattanell et al., 1978; Trias et ul., 1978; Ham et al., 1978; Britton et n l . , 1978; Terblanche et al., 1979; Ambtrup et al., 1980; Neuberger et al., 1980; Pietsch et al., 1981; Mahboubi et al., 1981; Horii et al., 1982; Helling and Wood, 1982; Shar and Kew, 1982) but also include unusual histologic types, such as eosinophilic hepatocellular carcinoma (Barrows et al., 1983), hepatic angiosarcoma (Monroe et al., 1981; Shi et aZ., 1981),combined bile duct and hepatocellular carcinomas (O’Sullivan, 1976), mixed hepatocellular carcinoma and sarcoma (Ladaga et al., 1979), mixed type hepatoblastoma (Meyer et al., 1974), and choiangiocarcinoma (Caggiano et uZ., 1980; Littlewood et al., 1980). These tumors varied in degree of anaplasia from well to poorly differentiated, and some had metastasized. Selective reporting of cases because they were taking OCs is always a possibility, and these reports cannot be considered strong evidence for a causal relationship

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

363

between the OCs and the hepatic neoplasms. Also, one study showed hepatocellular carcinomas from users of OCs to be more vascular than those from nonusers (Neuberger et al., 1980), which raises the possibility that users may develop symptoms and be diagnosed more readily than nonusers. However, this is not likely an important consideration, because few patients with hepatic carcinomas present with intraabdominal bleeding. Most of the carcinomas reported in users of OCs are in livers without evidence of cirrhosis, which suggests that alcoholism and hepatitis B virus (HBV), the usual factors of etiologic importance in many hepatic carcinomas, were not involved in their development. The American College of Surgeons survey of liver tumors in 15- to 45-year-olds in 749 U.S. hospitals (Vana et al., 1977) included 166 malignant lesions in women. Use of OCs was ascertained only for 90 (54%of these women, of which 49 (54%)had used OCs. In view of the high percentage with unknown use, this percentage does not seem unusually high, and this observation does not provide evidence for (or against) an association between OCs and liver cancer. In this series, the users of OCs tended to be younger than the nonusers, but this is not unexpected since only women of childbearing age use OCs, and these products were not used with sufficient frequency in the past for the older women in the series to have been as likely to have used them as the younger women. Goodman and Ishak (1982) reported results from a series of 128 cases of hepatocellular carcinoma in women in the files of the U.S. Armed Forces Institute of Pathology. Women under 40 tended to have fibrolamellar carcinoma, whereas older women more frequently had other types. This variant tends not to occur in conjunction with cirrhosis. The proportion of women under 40 who had used OCs was nearly the same for those with fibrolamellar and those with other histologic types, suggesting that oral contraceptives are not related specifically to fibrolamellar tumors. Individuals with this tumor tend to survive longer than persons with ordinary hepatocellular carcinoma (Craig et al., 1980), which may explain reports that young women with liver cancer who had used OCs had a better prognosis than nonusers with liver cancer (Neuberger et al., 1980). Three small case-control studies of malignant liver tumors and OCs have been reported (Table XXXVII). Twelve cases were included in the study of Henderson et al. (198313).All were between 18 and 39 years of age, had been born in the United States, and had developed their disease while living in Los Angeles County between 1975 and 1980. Eleven of the 12 cases were interviewed. For each interviewed

HOSS L. PRENTICE AND DAVID B. THOMAS

364

TABLE XXXVII RELATIVERISKS OF HEPATOCELLCLAH CARCINOMA IN RELATION TO USE OF ORAL ~

_

Source

CONTRACEITIVES: CASE-CONTROL STUDIES _ Category Number of subjects, of use caseslcontrols ~~

Henderson et al. (1983b) Neuherger et al (1986)' Fomian et al. (1986)

Summary RR

~

None Ever >5 years None Ever >8 years None Ever >8 years Ever Longest

1"/9 10 /13 6 I4 5I5.9d 17 /16.1d 8 /1.8" 4 168 15 179 3 14

RR estimate

(95% CI)" 1 .o

6.9 [0.7,64.0] 13.5 [1.2,152.2] 1 .o

1.5 (0.5,4.4) 7.2(2.0,25.7) 1.0 3.8 (1.0,14.6) 20.1 (2.3,175.7) 2.5 [1.1,5.5]e 10.0 [ 13.7,27.2]f

[ 1 Indicates confidence i n t e n d s calculated using the approximate methods described in the text. This case took no oral contraceptives but received injections of a hormone of unknown type for 9 months for regulation of menstrual periods. Excluding four cases with serologic evidence of prior HBV infection. Expected numbers of users and nonusers among cases, based on a total of 1,333 controls. p Value of x2 test for heterogeneih equal to 0.36. p Value of x2 test for heterogeneity equal to 0.70.

case two neighborhood controls were also interviewed. Ten of the 11 cases had used OCs and the other had received 9 months of hormone injections of undetermined type. By contrast, only 13 of the 22 controls had used OCs. I n addition, cases had used OCs for an average of 64 months whereas controls who had used OCs had used them for an average of 27 months. This difference in duration is unlikely to have occurred by chance (one sided p < 0.005). The tumors in the cases were of a variety of histologic types. Three had fibrolamellar carcinomas, four had ordinary hepatocellular carcinoma with varying degrees of differentiation, and one each had malignant giant cell, sclerosing duct forming, papillary, and microtrabecular carcinomas. In the study reported by Neuberger et al. (1986),the prior OC use of 26 women under age 50 with hepatocellular carcinoma who were referred to a liver unit in a London hospital was compared to that of 1,333 hospitalized women who had been included as controls in a case-control study of breast cancer in Britain. The relative risk of liver cancer was estimated to be 1.0 in women who ever used OCs and was

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

365

not increased in users of less than 5 years’ duration, but it was estimated to be 4.4 (1.5,12.8)in women who used OCs for 8 or more years. The increase in risk was limited to women without previous evidence of HBV infection. When the four cases with serologic evidence of prior HBV exposure were excluded, the relative risks for ever-users and users of 8 or more years were 1.5 (0.5,4.4)and 7.2 (2.0,25.7),respectively (Table XXXVII). Forman et al. (1986) identified all British women between the ages of 20 and 44 who died from liver cancer in the years 1979 through 1982. They were compared to a control group of British women of the same age who died during the same years of kidney or brain cancers, acute myeloid leukemia, or road accidents. Information on prior use of OCs was acertained from the records of the women’s general practitioners for 48‘of the 85 identified cases, and 147 of the 233 selected controls. On review of medical records, 24 of the cases were confirmed to have had hepatocellular carcinoma and 11 to have had cholangiocarcinoma. Five of the former were excluded because they had evidence of prior hepatitis, alcoholism, cirrhosis, or Downs syndrome, leaving only 19 hepatocellular carcinoma cases for analysis. No association was found between cholangiocarcinoma and OC use, the relative risk in ever-users and users of over 4 years’ duration being 0.3 and 0.9, respectively. The relative risk of hepatocellular carcinoma was found to be 3.8 (1.0J4.6) in women who ever used OCs, and 20.1 (2.3,175.7)in women who used them for 8 or more years (Table XXX-

VI I). Three of the women with hepatocellular carcinoma in the Forman

et al. (1986) study were also included in the study of Neuberger et al. (1986). Two were nonusers, and one had used OCs for more than 8 years. Removal of these subjects from the analyses would somewhat enhance the estimates of the relative risks. When the data from all three studies were combined, a summary relative risk of 2.5 was estimated for women who ever used OCs, and a value of 10.0 was estimated for long-term users (more than 5 or 8 years, depending on the study). All three of these studies are small, and, even when their data are combined, they provide imprecise measures of the association between OCs and liver cancer. All also have methodologic limitations which could have resulted in biased findings. However, the consistency of the results from these three investigations, which utilized different methods of case identification, control selection, and ascertainment of prior OC usage, and the uniformly strong associations observed suggest that primary hepatocellular carcinoma can be a complication of long-term use of OCs.

366

HOSS L. PHEN‘TICE AND DAVID B. THOMAS

Fortunately, this adverse effect, if real, is extremely rare. I n the two population-based studies (Henderson et al., 1983b; Forman et al., 1986), only 12 and 85 cases in women young enough to have been at risk of using OCs were identified in the entire population of Los Angeles County (over 7,000,000 people) and all of Britain in 6 and 4 years, respectively. No increase in mortality rates of liver cancer in young women has been observed in the United States, Australia, West Germany, or the h’etherlands (Forman et al., 1983). A small increase has, however, been reported in the United Kingdom (Forman et al., 1983), and incidence and mortality rates of liver cancer in young women should continue to be monitored in populations where OCs are widely used. In all three studies, risk was observed only in the absence of other factors known to be associated with liver cancer (i.e., hepatitis B virus, chronic hepatitis, cirrhosis, and alcoholism). This suggests that oral contraceptives may not interact with other hepatic carcinogens, and if they do not then they may not b e found to enhance measurably risk of liver cancer in parts of the world where HBV is endemic and hepatocellular carcinomas are common. Studies in high risk populations should nonetheless be conducted to determine whether OCs do enhance risk in the presence of hepatitis B virus and other risk factors for 1’iver cancer.

F. M A L I C N A ~MELANOMA T Estimates of the relative risk of malignant melanoma in women who ever used oral contraceptives from eight case-control studies are shown in Table XXXVIII. None differ significantly from unity, and the summary relative risk, based on data from seven of the studies is 1.0. The results from three cohort studies, summarized in Table XXXIX, are not consistent, with one showing a significant increase in risk and one showing a significant decrease in risk in OC users. The summary relative risk based on data from all three cohort studies is not significantly different from one, and is consistent with that from the casecontrol studies. Table XL shows relative risks in relation to duration of use from seven case-control studies. The results are inconsistent. Although the summary relative risk for long-term users, based on data from the largest use category in six of the studies, is 1.3, with a lower 95% confidence interval of 1.0, the study that could not be included in the calculation of this summary relative risk, which yielded a relative risk of 0.8 in users of 5 or more years, would result, if included, in a

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367

TABLE XXXVIII RELATIVE RISKSOF MALIGNANT MELANOMA IN WOMEN WHOEVER USED ORALCONTRACEETIVES: CASE-CONTROL STUDIES Caseslcontrols Source

Users

Nonusers

Beral et al. (1977) Adam et al. (1981b) Bain et al. (1982) Holly et al. (1983) Helmrich et al. (1984) Beral et al. (1984) Holman et al. (1984) Gallagher et al. (1985) Summary RR

22/33 391107 411807 181166 631270 2081413

13136 661214 8311687 391426 971370 791159

RR estimate (95%CI)" 1.8 [0.8,4.2] 1.1(0.7,1.8) 0.8 (0.5,1.3) 1.2 [0.6,1.8] 0.8 (0.5,1.3) 1.0 [0.5,1.9] 1.0 (0.6,1.6) 1.0b 1.0 [0.9,1.2]'

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. b Insufficient information published to include in calculation of summary relative risk. p Value of x2 test for heterogeneity equal to 0.69. Q

summary relative risk closer to unity. Table XLI shows that the results from two cohort studies regarding risk in relation to duration of use are also inconsistent, but the summary relative risk from these two investigations for users of 4 or more years is also close to one. The reasons for the inconsistent results among the various studies are unknown. TABLE XXXIX RELATIVE RISKSOF MALIGNANT MELANOMA IN WOMEN WHOEVER USED COMBINED ORALCONTRACEPTIVES: COHORTSTUDIES

Source Kay (1981) Ramcharan et al. (1981) Adam et al. (1981b) Summary RR

Category of use

Cases per 1000 person-years (number of cases)

RR estimate (95%CI)"

Never Ever Never Ever Never Ever Ever

11.0 (16) 15.0 (24) 0.08 (4) 0.28 (16) 0.17 (8) 0.04 (4) -

1.o 1.5 (0.7,2.9) 1.o 3.5 (1.4,g.O) 1.0 0.2 [0.1,0.8] 1.4 [0.8,2.3Ih

0 [ ] Indicates confidence intervals calculated using the approximate methods described in the text. b p Value of x* test for heterogeneity equal to 0.002.

368

ROSS L. PRENTICE AND DAVID B. THOMAS

TABLE XL RELATIVERISKS OF MALIGNANTMELANOMA IN RELATION TO DURATION OF USE OF ORAL CONTRACEPTIVES: CASE-CONTROL STUDIES Source Adam et at. (1981b)

Years of use

Number of subjects, caseslcontrols

Xone

661214 3/10 5112 2/13 12137 17/35 8711687 181231-

<0.5 0.5

1-2 3-4 5+ Bain et 02. (1982)

None

Holly et al. (1983)

2+ None

1-2 1-4 5-9 > 10

Helnirich et al. (1984)

None

Holman et at. (1984)

None <2

(1 1-4 5-9 2 10

601426

9/81 9153 9/32 971370 15/82 231106 11/49 5121 Not given

2-4 5+ Beral et at. (1984)

None

<1 1-4 5-9 10+

791159 29/73 95/201 56/103 28/36

Gallagher e t al. (1985)b

None

Not given

Summary RR

>1 1-4 5+ Longest

-

RR estimate (95% CI)”

p Value x2 test for trend

1.o 1.0[0.3,3.6] 1.4[0.5,4.0] 0.5 [0.1,2.3] 1.1 [0.5,2.1] 1.6(0.8,3.0) 1.0 0.8(0.5,1.5) 0.8(0.5,1.5) 1.0 1.0[0.5,2.1] 1.5[0.7,3.2] 2.1 [1.0,4.6] 1.0 0.7(0.4,1.3) 0.8(0.5,1.4) 0.8(0.4,1.7) 1.0(0.4,2.9) 1.0 0.7(0.4,1.2) 1.2(0.6,2.2) 1.1 (0.6,2.0) 1.o 0.8[0.5,1.3] 1.0[0.7,1.4] 1.1 [0.7,1.7] 1.6[0.9,2.7]

1.o

Not given

Not given

0.09

Not given

0.25

Not given

1 .o

0.9 0.8 1.3[1.0,1.7Ic

0.21

L1 [ ]Indicates confidence intervals calculated using the approximate methods described in the text. Insufficient information published to compute confidence limits and include in summary relative risk. p Value of xz test for heterogeneit). equal to 0.40.

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES

AND DISEASE

369

TABLE XLI RELATIVE RISKSOF MALIGNANT MELANOMA IN RELATION TO DURATION OF USE OF ORALCONTRACEPTIVES: COHORT STUDIES

Source Beral et al. (1977) Adam et al. (1981b)

Years of use None <4 24 None <4 124

Summary RR

24

Cases per 1000 person-years (number of cases)

0.176 (5) 0.241 (8) 0.295 (6) 0.17 (8) 0.06 (3) 0.02 (1)

-

RR estimate (95%CI)” 1.0 1.4 [0.4,4.2] 1.7 [0.5,5.5] 1.0 0.4 [0.1,1.3] 0.1 [0.01,0.9] 0.9 [0.3,2.5Ib ~

~~

[ ] Indicates confidence intervals calculated using the approximate methods described in the text. p Value for x2 test for heterogeneity equal to 0.03.

On balance, these discrepant findings do not provide convincing evidence that OCs enhance the risk of malignant melanoma. However, the two case-control studies in which analyses were performed to estimate the relative risk in users of 2 and 5 or more years’ duration, 10 or more years after initial exposure, both found modest elevations in relative risks of 2.3 (0.8,6.9)and 1.5 (1.0,2.1),respectively (Bain et al., 1982; Beral et al., 1984). These results suggest that risk might be elevated in long-term users after some latent period. Some studies have reported results separately for superficial spreading melanoma. Two case-control studies found trends of increasing risk of these lesions with duration of use (Holly et al., 1983; Holman et al., 1984), but a third one did not (Gallagher et al., 1985). Also, two studies (Beral et al., 1984; Holly et al., 1983) have shown relative risks of supervicial spreading type melanoma in users of 5 or more years’ duration after 10 and 12 years since initial exposure to be 1.6 (1.0,2.6)and 4.4. (2.0,9.7),respectively. Additional studies of OCs and malignant melanoma thus seem warranted. Particular attention should be given to assessing risk in long-term users after an appreciable period since initial exposure, and this should be done separately for superficial spreading melanomas as well as for other types and all types combined.

G. OTHERNEOPLASMS An increase in incidence rates of pituitary adenomas in women of childbearing age, but not in older women or in men, was noted in

370

ROSS L. PHENTICE AND DAVID B. THOMAS

Olmsted County, Minnesota, from 1935 to 1977 (Annegers et al., 1978). This led to the hypothesis that oral contraceptives might increase the risk of these tumors. However, subsequent case-control studies have not supported this hypothesis (Coulam et al., 1979; Wingrave et al., 1980; Shy et al., 1983). Women with prolactin-secreting tumors of the pituitary (prolactinomas) tend to have menstrual irregularity which is often treated with OCs or estrogens, and this has resulted in spuriously observed associations between prolactinomas and these therapeutically used preparations (Shy et al., 1983). A small, nonsignificant increase in risk of thyroid cancer was observed in the Walnut Creek cohort study (Ramcharan et al., 1981)but has not been confirmed by subsequent studies. This increased risk was probably a chance phenomenon. Women who took OCs after evacuation of a hydatidiform mole were reported by Stone et al. (1976) to subsequently develop prolific trophoblastic tumors more frequently than women who used other methods of contraception after a molar pregnancy. This observation was not confirmed in a small cohort study by Berkowitz et al. (1980), although the power of this study to detect a true increase in risk was low. Since prevention of pregnancies prevents hydatidiform moles and hence also invasive trophoblastic disease, OCs, like other successful methods of birth control, would be expected to have a net protective effect against this neoplasm when used to limit family size. A single population-based case-control study of carcinomas of the colon and rectum in Australia (Potter and McMichael, 1983)showed a reduced risk of these neoplasms in users of OCs. The risk of colon cancer declined with duration of use. None of the three large cohort studies (Vessey et al., 1976; RCGP, 1974; Ramcharan et al., 1981) have reported an alteration in risk of this relatively common condition, and this isolated finding must be viewed with skepticism pending independent confirmation. A protective effect of OCs against benign breast diseases has been well documented in a review by Thomas (1978), and in subsequent investigations (LiVolsi et al., 1978; Brinton et al., 1981; Ramcharan et al., 1981; Pastides et al., 1983; Hsieh et al., 1984; Hislop and Threlhll, 1984). This effect has generally been observed for both fibroadenomas and fibrocystic disease. Although one recent investigation found an increase in risk of the latter condition in postmenopausal women (Berkowitz et al., 1984), the bulk of the evidence is at variance with this largely isolated finding. Women with fibrocystic breast disease are at increased risk of developing breast cancer, particularly if their lesions are characterized by

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

371

cellular proliferation and atypia (Hutchinson et al., 1980; Page et al., 1985). Oral contraceptives have not been shown to protect against breast cancer. LiVolsi et al. (1978) reported no reduction in risk of fibrocystic disease with atypical histologic features, and suggested that this explained the apparent contradiction. However, this finding was not confirmed in two subsequent investigations (Pastides et al., 1983; Hsieh et al., 1984). It is possible that prior atypical benign lesions account for such a small proportion of all breast cancers that a reduction in their incidence results in a reduction in risk of breast cancer that is too small to be detected by available epidemiologic methods. Reduced incidence rates of retention cysts of the ovary were observed in OC users in both British cohort studies (Vessey et al., 1976; RCGP, 1974) and in a case-control study (Ory et al., 1974). Since OCs inhibit ovulation, these are not surprising findings and almost certainly represent a true protective effect. Risks of other benign ovarian tumors were not altered in the two British cohort investigations or in the Walnut Creek cohort study (Ramcharan et al., 1981). Risk of uterine fibroids was found to be increased in the Walnut Creek investigation, although the authors note that this could have resulted from better detection in OC users than nonusers. No increased risk of these tumors was found in the RCGP study (1974), and a reduced risk with duration of use was found in the Oxford Family Planning Association study (Ross et al., 1986). VI. Oral Contraceptives and Other Diseases

Since oral contraceptives have been used by many millions of women in all parts of the world for over two decades, virtually all known diseases have developed in women who have used these steroid preparations, and many published case reports undoubtedly represent chance occurrences. Also, the development of all diseases and conditions requiring medical treatment is monitored in the three large cohort studies (RCGP, 1974; Vessey et al., 1976; Ramcharan et al., 1981), and some will be found by chance alone to have occurred with altered frequency in users of OCs. Because of these considerations, the diseases and conditions included in this section are restricted to those with increased or decreased incidence rates in at least two cohort studies, or in at least one case-control study that was designed specifically to investigate, among other things, whether OCs alter the risk of the disease in question. Both British cohort studies have shown a reduction in risk of various

372

ROSS L. PHENTICE AND DAVID B. THOMAS

menstrual disorders other than amenorrhea and an increase in risk of amenorrhea or scanty menstrual flow in users of OCs. It is thus not surprising that a reduction in incidence of iron deficiency anemia has also been observed in all three cohort studies (RCGP, 1974; Vessey et ul., 1976; Ramcharan et al., 1981).The amenorrhea can persist for up to 2 years or more after cessation of use of OCs (Furuhjelm and Carlstrom, 1973), but risk of permanent infertility does not appear to be enhanced in former users (RCGP, 1974; Vessey et al., 1978). The RCGP study showed OC use to be associated with reduction in risk of rheumatoid arthritis (Wingrave and Kay, 1978) and three classes of thyroid disease, euthyroid swelling, thyrotoxicosis, and myxoedema (Frank and Kay, 1978); Frank and Kay hypothesized that these findings are a result of suppression of autoimmunity by the contraceptive steroids. Hoover et al. (1978), however, reported an increase in rates of hospitalization for hyperthyroidism in OC users, and the alleged protective efyect of these preparations against rheumatoid arthritis has not been confirmed by others. Increased incidence rates of hay fever and allergic rhinitis were observed in users of OCs in all three cohort studies, although not significantly so in the Walnut Creek investigation (Ramcharan e t a?., 1981). Risk was related to estrogen dose in the RCGP study (RCGP, 1974). Rates of conjunctivitis and diseases of the eyelid were also slightly increased in both the RCGP and Walnut Creek studies, and rates of eczema were elevated in OC users in both British cohort investigations. Since none of these allergic phenomenon are life threatening, it is possible that their observed associations with OC use are due to more complete reporting of these conditions by users than nonusers. A true effect of the steroids in oral contraceptives on the immune system cannot currently be ruled out as an explanation, however. Increased rates of regional enteritis and ulcerative colitis in users of oral contraceptives have been reported from both British cohort studies (RCGP, 1974; Vessey et al., 1986b) and from the Walnut Creek study (Ramcharan et al., 1981);small increases in gastritis, duodenitis, and appendicitis, have been noted in the latter investigation and the RCGP study, although not by Vessey et al. (1976).These observations may not represent causal associations and require further investigation. Gallbladder disease (cholecystitis and cholelithiasis) has been related to short-term use of OCs in a large hospital-based case-control study (Boston Collaborative Drug Surveillance Program, 1973) and in both British cohort studies (Layde et al., 1982a; Kay, 1984). However,

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

373

risk was not increased in long-term users and, in the RCGP study, rates increased with duration until the third year of use, and then declined so that they were actually lower in users than nonusers after 7 years of use (Kay, 1984). This has been interpreted as indicating that OCs accelerate the development of gallbladder disease in women with asymptomatic preexisting disease. Dalen and Westerholm (1974) found that women who developed jaundice while taking OCs tended more frequently than users without jaundice to have a prior history of pruritus and jaundice during pregnancy, hepatitis, symptoms of gallstones, and cholecystectomy, and a family history of these conditions in their mothers or sisters. The bile of women taking OCs has been shown by Bennion et al. (1976) to be more saturated with cholesterol than the bile of nonusers, and it is likely that OCs either enhance the formation of gallstones in susceptible individuals, or promote growth of preexisting stones. Women who have taken OCs have been shown in all three cohort studies to be at increased risk of cervical erosion, vaginal discharge, or infection of the pelvic organs. It is likely that associations between OCs and the latter two conditions are due at least in part to differences in the sexual behavior of users and nonusers. An increased risk of urinary tract infections was observed in the RCGP study (1974) and the Walnut Creek investigation (Takahashi and Loveland, 1974), but not in a large community-based prevalance study (Evans et al., 1978); this purported adverse effect is probably also explainable by differences in the sexual behavior of women who chose the pill over other methods of contraception. All three cohort studies showed an increase in rates of migraine headaches in OC users, although not significantly so in the RCGP investigation (1974). Misclassification of headaches associated with OC-induced hypertension is one possible explanation. Another is that these observations may be a result of more frequent referral of these conditions for treatment in OC users than nonusers. However, the uniformity of the results from all three studies is evidence that the association may be causal, and additional investigations of this possibility are warranted. An increase in rates of referral for depression was reported in OC users in the Walnut Creek study (Ramcharan et al., 1981), and an increased risk of hospitalization for mental or emotional illnesses was observed in a cohort study in Boston (Hoover et al., 1978). In addition, Vessey et al. (1976) and Ramcharan et al. (1981) reported increased rates of self-poisoning and suicides (as well as homicides) in pill users, and Kay (1984) reported increased rates of suicides. How-

374

ROSS I,. PRENTICE AND DAVID B. THOMAS

ever, no increase in risk of depression in OC users was found by Vessey et al. (1976); in a more recent report, Vessey et a2. (1985) indicate that rates of hospitalization for both psychotic and nonpsychotic disorders were similar in users and nonusers of OCs. Also, the increases in rates of depression and suicides in the Walnut Creek Study were not statistically significant ( p > 0.05);no increase in risk of depression with duration of use was observed in the Boston study; and in the RCGP study (Kay, 1984) although rates of neurotic depression were significantly increased in current users, they were significantly decreased in former users. Furthermore, a double blind, crossover trial of 45 women (Leeton, 1973), in which all subjects were administered a questionnaire designed to provide an index of depression, showed no significant difference in index scores between women on OCs and women on placebos and showed no relationship between index scores and the progestogenic effect of the pill on vaginal smears. The most likely explanation for these observed mental and social problems is that women at risk of these conditions tend more frequently to choose the pill than other methods of contraception. VII. Risk-Benefit Summary and Future Research Needs

The risk-benefit analysis presented here is restricted to estimated OC impact on total mortality, in order to avoid such issues as the comparative seriousness and comparative impact of various morbidity categories or the possiblity that two or more diseases may arise in the same individual. One of the most meaningful and straightforward risk-benefit summaries of OC impact on societal mortality is given in Section I11 (Table I). Specifically, on the basis of the follow-up of sizeable British and American cohorts of women, mostly initially in the age range 20-49, for an average of about one decade from the late 1960s to the late 1970s, it was estimated that women who were everusers of OCs had a 20% higher overall mortality (95% confidence interval 1.0 to 1.4 for relative risk) than did never-users. Comparability of the ever- and never-user groups was attempted in each of the studies by standardizing mortality rates in respect to age, cigarette smoking habits, parity, and certain other factors. Considering that we are dealing with total mortality an increase of this magnitude seems noteworthy indeed. Some previous reviewers (Ory et al., 1980) have asserted “we can conclude with some assurance that for most healthy, young women, the benefits of oral contraceptive use continue to outweigh the risks.” Subsequent paragraphs will attempt to assess this assertion in respect to overall mortality. As a

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

375

first step toward this end it seems noteworthy that in the largest of the cohort studies (RCGP, 1981a), involving 46,000 women, there were only three deaths attributed to complications of pregnancy, childbirth, and puerperium (ICD 630-678), and two of the deaths were among ever-users of OCs. In contrast, 55 of the 156 deaths in ever-users were of circulatory system causes as compared to only 10 of 93 among never-users. Also these previous reviewers evidently did not acknowledge any altered disease risks among former OC users in their assessment. The total mortality summary from Table I does not address the risks and benefits as a function of individual characteristics, such as age, cigarette smoking habits, or predisposition to various diseases, or as a function of OC usage pattern and formulation characteristics. To address these topics various relative risk estimates will be extracted from the preceding tables and applied to site-specific mortality data from the United States for each of a range of patterns and characteristics. Toward this end relative risk estimates for cardiovascular disease (Section IV) and cancer (Section V) will first be summarized. Table XLII displays relative risk estimates and confidence intervals taken from the previous tables, for both current and former OC use. For cardiovascular diseases the cohort study estimates generally apply to all incident disease, whereas nonfatal disease summary statistics were selected for the case-control studies since such nonfatal estimates tended to be more accurately and precisely determined. As an exception, the subarachnoid hemorrhage relative risk estimate (1.5) for former OC usage applies to fatal disease, since no corresponding nonfatal disease estimate was available. Note the excellent agreement between cardiovascular disease relative risk estimates from the two types of study. The sole exception occurs for nonhemorrhagic stroke where the case-control estimate (7.2) is notceably higher than the cohort estimate (3.8), possibly due to different relative risks for fatal and nonfatal disease or possibly due to other study eligibility requirements (e.g., with respect to predisposing factors) in the case-control studies. Table XLIII lists the relative risk estimates selected for the subsequent mortality risk-benefit analysis. Specifically, the cohort study relative risk estimates are used for total cerebrovascular disease, while myocardial infarction relative risk estimates are permitted to depend on the duration of current or former OC use in the manner given in Table IX. The myocardial infarction relative risk estimates associated with former OC use are rather important to the risk-benefit analysis, and it should be remembered that these estimates are based

SUMMARY HELA'I'IVE:

TABLE XLII ESTIMATES ?\NDAPPROXIMATECONFIDENCE INrERVALS FOR VARIOUS CARI)IOVASCULAH DISEASE A N U CANCER CATEGORIES

HISK

Current OC use

Former OC use

Disease category (ICL) code)

Cohort studies

Case-con trol studies

Cohort studies

All cerebrovascular disease (430-438) S uharachnoid hemorrhage (430) Nonhemorrhagic stroke (432-438) Myocardial infarction (410) Peripheral arterial disease (440-448) Venous thromboemholism (450-453) Idiopathic venous thromboembolism (450-453)

2.9 [2.0,4.1] 2.0 [1.1,3.61 3.8 [2.4,6.11 1.9 (1.1,3.3) 1.6 [1.3,2.1] 2.7 [2.0,3.8Iid 6.8 [3.5,13.2]

2.3 [1.6,3.11 2.0 [1.2,3.4] 7.2 [3.8,13.71 2.2 [1.6,2.9]

1.8 [1.3,2.6] 2.1 [1.1,3.4] 1.9 [1.2,3.0] 1.1 (0.6,1.9)

Ever OC use, case-control studies Endometrial cancer Breast cancer Ovarian cancer (Invasive) cervical cancer This estimate arises from RCGP (1978).

0.5 [0.4,0.6] 1.0 [0.9,1.01 0.6 [0.5,0.7] 1.3 [1.1,1.5]

5.8 [4.3,7.9]

Case-control studies

1.5 [1.0,2.1] 1.1 [0.9,1.31

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

377

TABLE XLIII ASSUMEDRELATIVERISKESTIMATES FOR MORTALITY RISK-BENEFIT ANALYSIS

Cause of death category (ICD) code)

All cerebrovascular disease (430-438) Myocardial infarction (410) Peripheral arterial disease (440-448) Venous thromboembolism (450-453) Endometrial cancer (181, 182) Breast cancer (174) Ovarian cancer (180) Cervical cancer (180) Pregnancy, childbirth, and the puerperiurn (630-678) All other diseases

Duration of OC use (years)

1-4 5-9 10+ Any

Any Any 1-4 5t 1-4 5+ Any

Relative risk estimate Current OC use

Former OC use

2.9

1.8

1.5 2.6 4.4 1.6

1.0 1.4 2.3 1.0

2.7

1.0

0.5 1.0 0.7 0.4 1.1 1.6" 0.2

0.5 1.0 0.7 0.4 1.1 1.6 1.0

1.0

1.0

a 1.6 is the summary RR from Table XXXV, excluding data from LaVecchia et al. (1986),which appeared after the subsequent calculations were completed.

on a single study (Slone et al., 1981). The RCGP (1978) relative risk estimate (2.7) was chosen for venous thromboembolism. The risk of peripheral arterial disease and venous thromboembolism is taken to be the same in former OC users as in never-users. The relative risk estimates for both ovarian and cervical cancer are allowed to depend on duration of OC use in a manner suggested by Tables XXIII and XXXV, respectively. The relative risk estimates just described were taken to apply the mortality data, even though such were largely developed based on incidence data. On the whole the previous sections do not suggest important differences between the OC-associated relative risks for incident as compared to fatal disease, and the incident disease relative risks are much more precisely estimated. Note, however, that for some disease categories, particularly certain cancers, OC-induced changes in the mortality relative risks will be expected to Iag the corresponding incidence relative risks by some years. The final rows of Table XLIII specifiy a relative risk estimate of 0.2

378

ROSS L. PRENTICE AND DAVID B. THOMAS

for mortality due to complications of pregnancy, childbirth, and the puerperium among OC users, with no alteration in risk among former users. Ory et al. (1983) list an 11%annual pregnancy rate among U.S. women of childbearing age and a 2.4%annual contraceptive “failure” rate among current OC users. Assuming that 12.4%of women of childbearing age are current OC users (see below) then leads to an estimated pregnancy rate of 12.2 among all women of childbearing age who are not current OC users, hence the assumed pregnancy relative risk estimate of 2.4/12.2 = 0.2 among current OC users. Finally, in order to link this number to mortality data it is necessary to assume that, for a pregnant woman, the risk of death from complications of pregnancy, childbirth, and the puerperium is unrelated to OC usage pattern prior to pregnancy. Note that this relative risk estimate (0.2) involves a comparison of maternal mortality among OC users to that among nonusers having the mixture of other contraceptive practices (among non-OC users) prevailing in the United States. Mortality rates for all other cause of death categories are assumed (Table XLIII) to be unaltered b y current or former OC use. This category includes disease categories such as liver cancer for which an adverse OC effect is suspected. These diseases are sufficiently rare in the age range to be considered that acknowledgment of such an adverse effect would have little influence on subsequent calculations. The studies on which the relative risk estimates described above were developed mainly involved disease arising from the late 1960s to the early 1980s. Hence they will be applied to mortality data for 1975, the approximate midpoint of such a time period. Table XLIV gives age-specific mortality rates, for ages 20-59, for all U.S. females in 1975 for each of the cause of death categories of the preceding table. These numbers, in conjunction with data on frequency of current and former OC use, classified by duration of use, can be used to estimate an agespecific mortality rate for never-users of OCs and then for each of the desired OC use categories upon applying the relative risks of Table XLIII to the never-user mortality rate. Table XLV shows “estimated” frequencies of various OC usage and duration categories by age group. These frequencies were adapted from control group frequencies in Slone et al. (1981), based on data collected in 1976 and 1979, by assuming that the duration of use among current users was independent of age and by assuming that the duration of use among former users was independent of age within age groups 25-39 and 40-49, respectively. Furthermore, the frequencies given in Table XLV were extrapolated to the age period 20-24 by assuming the same frequencies of current OC use as for the 25-29 age

TABLE XLIV AGE-SPECIFIC MORTALITYRATESFOR ALL U.S. FEMALES IN 1975 ~

Annual mortality rate per 100,000 women,O by age category Cause of death (ICD code) All cerebrovascular disease (430-438) Myocardial infarction (410) Peripheral arterial disease (440-448) Venous thromboembolism (450-453) Endometrial cancer (181, 182) Breast cancer (174) Ovarian cancer (183) Cervical cancer (1980) Pregnancy, childbirth, and the puerperium (630-678) All other causes of death Total

20-24

25-29

30-34

35-39

40-44

45-49

50-54

55-59

1.5 0.2 0.3 0.8 0.1 0.2 0.4 0.2 1.0

2.8 0.5 0.5 0.9 0.2 1.6 0.5 1.0 1.2

4.7 2.0 1.0 1.6 0.3 5.4 0.9 2.1 1.o

8.6 5.9 1.5 1.7 0.7 12.4 2.3 3.9 0.9

15.2 14.2 2.1 2.9 1.3 24.8 5.5 6.6 0.3

23.4 29.4 3.4 4.2 2.8 42.4 12.4 8.8 0.0

37.9 58.0 6.0 6.2 5.2 58.2 18.8 10.4 0.0

58.9 109.7 10.5 8.4 9.7 73.8 24.4 11.4 0.0

62.5 67.2

65.3 74.5

78.8 97.8

108.3 146.2

164.3 237.2

239.5 366.1

343.5 544.2

514.5 821.3

Source: National Center for Health Statistics, Vital Statistics of the United States, 1975, Vol. 2, Mortality, Part A (1977).

380

ROSS L. PRENTICE AND DAVID B. THOMAS

TABLE XL\' ESTIMATED FREQUENCIES (%) OF CURRENT AND FORMER OC USEBY AGEAND DURATION OF USE"

OC usage, duration

25-29

30-34

35-39

40-44

45-49

38.2 2.3 2.0 1.6 42.8 11.2 1.9

57.0 0.5 0.4 0.4 31.4 7.0 3.5

73.3 0.3 0.2 0.2 19.5 4.4 2.2

~

~~

Never-user Current user, 1-5 years Current user, 5-9 years Current user, 101 years Former user, 1-5 years Former user, 5-9 years Former user, 10+ years

18.2 9.9 8.3 6.8 43.5 11.4 2.0

34.2 3.0 2.5 2.0 44.6 11.7 2.0

" Adapted from control group data in Slone et al. (1981).

group and by combining the fornier use frequencies in the 25-29 age group with the nonuse frequencies to give a never-use frequency of 75.1%for the 20-25 age range. These frequencies were also extrapolated to the age intervals 50-54 and 55-59 b y assuming no current OC usage and a common distribution of former use frequencies to that for ages 45-49. These estimates may involve lower current OC use and somewhat higher former use frequencies than likely obtained in 1975. For example, surveys of U.S. women by the National Center for Health Statistics (Ford, 1978) gave estimates of current OC usage in the age range 15-29 of 37.6% in 1973 and 35.1% in 1976, as compared to 25.0% in Table XLV. Corresponding estimates for the age range 30-44 were 14.8% in 1973 and 11.8% in 1976. These survey figures were restricted, however, to women who were currently or previously married or who were never married but had offspring living in the household. The control group in the 1976 survey of American Nurses (Rosenberg et aZ., 1980) reported current OC usage of 12.4%among women less than 45 years of age and 8.1%among older women. The estimated frequencies of foimer OC usage in the two age groups were 22.8% and 17.9%, respectively, considerably lower than in Table XLV. The Nurses survey results were restricted, however, to premenopausal women. The estimates of OC usage given in Slone et al. (1981) were, in part, selected because they provide a detailed classification by age and because suitably classified data on duration of use among both current and former OC users is given. Undoubtedly more precise data on OC usage patterns could be gleaned froni the literature, but subsequent

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

381

calculations are expected to be robust to moderate variations in the frequencies in Table XLV. In fact, these frequencies are used only to estimate age-specific mortality rates among never-users. As an example of an estimated mortality rate among never-users of OCs, consider myocardial infarction incidence among women aged 35-39. The population annual mortality rate of 5.9 per 100,000 (Table XLIV) can be written in terms of the never-user mortality rate I as

5.9 = Z(0.382 + (1.5)(0.023)+ (2.6)(0.020)+ (4.4)(0.016) + (1.0)(0.428)+ (1.4)(0,112)+ (2.3)(0.019)} using the estimated OC frequencies in Table XLV and the corresponding relative risk estimates from Table XLIII. This gives I = 5.1 as the estimated annual myocardial infarction mortality rate per 100,000 women among never-users. The corresponding estimated annual myocardial infarction mortality rate among women in the same age group who are current OC users with duration of use of 10 years or more, for example, is then 4.4(5.1) = 22.2 per 100,000 women. Table XLVI uses this type of calculation to derive estimated total mortality relative risks over a 20-year period following the initiation of OC use, as a function of various ages of starting OC use and of duration of use. The information reviewed throughout this report is largely based on OC usage between the early 1960s and the early 1980s. Hence formal calculations pertaining to possible OC-induced risks TABLE XLVI “ESTIMATED” OC-INDUCED PERCENTAGE INCREASEIN CUMULATIVE MORTALITYRATE OVER THE 80-YEAR PERIOD FOLLOWING INITIATION OF OC USE,AS A FUNCTION OF OC USAGE CHARACTERISTICS Age at initiation of OC use Duration of OC use (years)

20

25

30

35

40

2.5 7.5 12.5 17.5

2.0“ 3.6 6.6 10.4

2.5 5.3 10.7 16.7

3.1 6.7 14.7

3.4 8.1

3.7

Estimated 20-year mortality rate (%) among OC never-users

1.89

6.37

9.70

2.72

4.16

2.0 is the estimated percentage increase in cumulative mortality for a woman in this category over the age range 20-40. The corresponding estimated cumulative mortality rate itself is, using the final row of the table, 1.89(1 + 2.0/100) = 1.93%.

382

HOSS L. I’RENTICE AND DAVID H . TIIOMAS

and benefits beyond 20 years from initiation of use do not seem warranted at this time. As an example of the Table XLVI calculations consider the entry for OC use that began at age 20 and continued for a 12.5-year period. The numerator of the relative risk for this entry in Table XLVI is then the sum over each of the cause of death categories in Table XLIV of the estimated mortality rates for a current user of 1-4 years’ duration aged 20-24 during the first 5 years, plus the sum of the mortality rates for a current user of 5-9 years’ duration aged 25-29 during the next 5 years, pluz ”ie sum of mortality rates for a current user of 10+ years’ duration aged 30-34 during the next 2.5 years, plus the sum of mortality rates for a former user of 10+ years’ duration aged 30-34 during the subsequent 2.5 years, plus the sum of mortality rates for a former user of 10+ years’ duration aged 35-39 over the final 5 years. The denominator is comprised of the same summation but for OC never-users throughout. From Table XLVI it can be noted that the total mortality rate is “estimated” to increase at each age at initiation and duration of OC use combination. For example, a woman that begins using OCs at age 25 and continues for 17.5 years has an estimated 16.7% increase in mortality rate over the age range 25-45. The absolute mortality rate for such a women is estimated as 2.7211.167) = 3.17% as compared to 2.72% for a corresponding OC never-user using the final row of Table XLVI. The percentage increases listed in Table XLVI seem supportive of, but perhaps somewhat smaller than, the 20% overall mortality rate increase among OC ever-users estimated by the three cohort studies (Table I). Perhaps the least well established of our relative risk assumptions concerns the postulated increase in MI risk among women who had formerly used OCs for 5 or more years (Table XLIII). Table XLVII repeats the risk-benefit calculation of Table XLVI under the assumption of no elevation in MI mortality among former OC users. The estimated percentage increases in total mortality rate are somewhat reduced among women with substantial duration of OC use (7.5 or 12.5 years) but are not much changed otherwise. In fact certain entries are even slightly increased in view of corresponding larger estimated disease-specific mortality rates among never-users. Subsequent calculations revert to the relative risk assumptions of Table XLIII. Risks and benefits may be quite different among women who are at relatively low risk for the diseases that appear to be OC related. Table XLVIII gives similar calculations to those given in Table XLVI but separately for nonsmokers and smokers, Nonsmokers have lower than average mortality from myocardial infarction, stroke, and cervical car-

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

383

TABLE XLVII “ESTIMATED” OC-INDUCED PERCENTAGE INCREASEIN CUMULATIVE MORTALITYRATE OVER THE %)-YEAR PERIOD FOLLOWING INITIATION OF OF OC USAGE CHARACTERISTICS AND WITH OC USE,AS A FUNCTION RELATIVERISKASSUMPTIONS MODIFIEDTO EXCLUDE ANY INCREASEIN MYOCARDIAL INFARCTIONMORTALITY AMONG FORMER OC USERS Age at initiation of OC use Duration of OC use (years)

20

25

30

35

40

2.5 7.5 12.5 17.5

2.0 2.8 4.7 9.7

2.5 3.9 7.1 15.6

3.1 4.7 9.7

3.4 5.4

3.7

Estimated 20-year mortality rate (%) among OC never-users

1.89

2.73

4.17

6.39

9.75

cinoma and hence can be expected to have a more favorable riskbenefit picture in respect to OC use than do smokers. The calculations of Table XLVIII assume a stroke relative risk estimate of 1.7 associated with current cigarette smoking, a number obtained by combining thrombotic and hemorrhagic stroke results in the Collaborative Group for the Study of Stroke in Young Women (1975) report. Similarly a myocardial infarction relative risk estimate of 2.8 associated with cigarette smoking was used, as was obtained in the Nurses Health Study (Rosenberg et al., 1980). The cigarette smoking relative risk estimate for cervical cancer was taken to be 1.7, as can be obtained from a Walnut Creek cohort report (Peritz et al., 1977), while that for “all other causes of mortality” was somewhat arbitrarily taken to be 1.2 in order to acknowledge the dependence of mortality rates from a number of other cancers, notably lung cancer, on cigarette smoking. Cigarette smoking relative risks for the other causes of death listed in Table XLIII were all taken to be 1.0, and a multiplicative relative risk model between OC usage pattern and cigarette smoking was assumed. Finally the “current” cigarette smoking rate was assumed to be 38%, as was the case for the control group in Rosenberg et al. (1980), in each age category. OC usage frequencies were taken to be as given in Table XLV for both nonsmokers and smokers. As an example of the calculations underlying Table XLVIII the annual myocardial infarction mortality rate for nonsmoking, OC never-users in the age range 35-39 is estimated to be 3.0 per 100,000 women, as com-

TABLE XLVIII OC-INDUCEI) PERCENTACE INCREASE IN CUMULATIVE MORTALITY RATE OVER THE %)-YEAR PERIOD INITIATION OF O c USE, AS A FUNCTION OF O c USAGE CHARACTERISTICS AND CIGARETTE SMOKING HABITS

“ESTIMATED” FOLLOWING

Nonsmokers, age at initiation of O C use

Cigarette smokers, age at initiation of O C use

Duration of OC use (years)

20

25

30

35

40

20

25

30

35

40

2.5 7.5 12.5 17.5

1.5 2.6 4.9 7.8

2.0 3.9 7.8 12.3

2.4 4.9 10.6

2.6 5.8

2.9

2.6 4.8 9.0 13.8

3.3 7.1 14.6 22.4

4.0 9.2 20.2

4.4 11.1

4.8

Estimated 20-year mortality rate (%) among O C never-users

1.74

5.76

8.70

2.50

3.79

2.12

3.08

4.76

7.36

11.34

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

385

pared to the national rate of 5.9. The corresponding annual rate for a smoker who is a current OC user of 10 years’ or more duration is then estimated to be (4.4)(2.8)(3.0)= 37.0 per 100,000 women. Table XLVIII indicates that under the assumptions listed the estimated overaIl mortality risks associated with each OC usage pattern are greater than unity for both cigarette smokers and nonsmokers. The percentage increase in 20-year mortality rate associated with a specific OC pattern is uniformly smaller for nonsmokers than for smokers by a factor of approximately 0.5, but the estimated increases associated with long-term OC use are appreciable even for nonsmokers. A further mortality calculation considers both cigarette smoking and hypertension in evaluating the risks for benefits of OC use (Table XLIX). For this purpose a relative risk model that is multiplicative in the three factors was assumed. Hypertension was assumed to convey a relative risk estimate of 6.5 for stroke, as is obtained by combining thrombotic and hemorrhagic stroke data in the Collaborative Group (1975) report while also comparing the combined moderate and severe hypertension categories to combined normal or borderline blood pressure categories. Hypertension was also assumed to be associated with a relative risk estimate of 3.8 for myocardial infarction (Rosenberg et al., 1980). Hypertension relative risk for all other categories were taken to be 1.0, and the hypertension prevalence rate was estimated to by 7.6%,equal to the control group estimate in Rosenberg et al. (1980), for all age categories. Finally the OC usage patterns were assumed independent of both cigarette smoking and hypertension. This assumption is undoubtedly somewhat inaccurate but moderate departures from such independence will have a modest impact on the entries of Table XLIX. Note from Table XLIX that, under the assumptions listed above, each OC usage pattern is associated with an estimated mortality rate increase in each cigarette smoking and hypertension category. The percentage increase is comparatively small for nonsmokers who are not hypertensive, ranging from 0.9 to 9.8%as function of age at initiation of, and duration of, OC use. As previously, cigarette smoking noticeably increases such estimated percentage increases, at a given hypertension status. Under the assumptions made, hypertensives have very much increased mortality consequences associated with OC use. Among hypertensive smokers even short-term use (2.5 years) is estimated to increase overall (20 years) mortality by 11-16% depending on age at initiation of OC use, while longer term OC use (20 years) is associated with an estimated increase in the range of 50%. One can readily use Table XLIX to estimate the excess mortality asso-

Nonhypertensive

Duration of OC use (years)

2.5 7.5 12.5 17.5 Estimated 20-year mortality rate (%) among OC: never-users

Nonsmoker, ago at initiation of OC use

Cigarette smoker, agc’ ;kt initiation of OC iisr

20

25

30

35

40

20

25

30

35

40

0.9

1.2 2.8 6.1 9.8

1.4 3.5 8.3

1.5 4.1

1.7

1.7 3.7 7.1 11.0

2.2 5.5 11.8 18.2

2.7 7.2 16.5

2.9 8.7

3.2

1.9 3.7 6.2 1.73

2.46

3.72

5.62

8.46

7.08

10.83

2.09

3.01

4.61

Hypertensive Nonsmoker, age at initiation of OC use

__

Cigarette smoker, age at initiation of O C use

Duration of OC u s e (years)

20

25

30

35

40

20

25

30

35

40

2.5 7.5 12.5 17.5

8.1 11.1 17.5 25.8

10.2 15.0 25.4 37.9

11.9 18.4 33.0

13.1 21.4

14.0

11.1 16.4 27.9 41.4

13.4 21.9 40.7 60.8

15.0 26.4 52.3

15.9 30.3

16.4

Estimated 20-year mortality rate (%) among OC never-users

1.95

2.92

4.64

10.76

17.56

7.35

11.53

2.51

3.94

6.52

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

387

ciated with OC use. For example, a hypertensive smoker who begins OC use at age 30 and continues for 12.5 years has an estimated excess cumulative mortality of 6.52(0.523)= 3.41%,whereas a normotensive nonsmoker with the same OC pattern has an estimated excess of only

3.72(0.083) = 0.31%. The calculations of Tables XLVIII and XLIX were repeated while assuming no increase in MI mortality among former OC users. The estimated percentage increases in total mortality were positive for every OC usage pattern regardless of smoking or hypertension status. The changes from Tables XLVIII and XLIX parallel those between Tables XLVI and XLVII. The above calculations provide a strong support for previous recommendations that OC use should be avoided by women with significant cardiovascular disease risk factors and by cigarette smokers, particularly older cigarette smokers. These calculations do not support the contention (Ory et al., 1980) that the benefits of OC use exceed the risks for most young healthy women, at least with respect to total mortality. The previous reviewers’ evaluation has already been criticized for not acknowledging adverse or beneficial effects associated with former OC use. An additional criticism pertains to the use of maternal mortality data in that earlier review. Specifically Ory et al. (1980) contrasted an OC-induced annual cardiovascular disease mortality of 3.7 per 100,000 women in the United States to a maternal mortality of 20.6 per 100,000 live births, noting that the latter is five times the former. The difficulty with this comparison is that the live birth rate is only about 6.6%per year among women of childbearing age so that a more appropriate comparison in respect to overall mortality is a cardiovascular mortality of 3.7 per 100,000 versus an annual maternal mortality of 20.6(0.066) = 1.4 per 100,000 (cf. Table XLIV). The preceding analysis improved upon this comparison by bringing in OC association with a range of other diseases and by acknowledging a small maternal mortality rate among OC users. There are several qualifications of the risk-benefit summary given above. First, it was intended to provide an overall summary of data pertaining to OC use during the 1960s and 1970s. OC dosage and formation, and trends in dosage and formulation during and after such a time period, are not acknowledged. Certainly the data summarized previously suggest that cardiovascular disease risks can be reduced by the use of OC preparations with a relatively low progestogen content, though some of the apparent benefits for certain cancers may also be reduced.

388

ROSS L. PRENTICE AND DAVID H . THOMAS

As an illustration of possible mortality implications for a low progestogen preparation the calculations of Tables XLVI were repeated with the difference from unity of each of the relative risk estimates of Table XLIII except maternal mortality reduced by 60%. Such a reduction was suggested by the fact that the ratio of the difference from unity of the estimated total arterial disease relative risk in the Royal College of General Practitioners cohort study for the 50 pg ethinyl estradiol and 1 mg norethindrone acetate (Kay, 1982) to that for all preparations JRCCP, 1983) is 0.40. The maternal mortality relative risk estimate was unaltered from Table XLIII. Table L shows results of these calculations. Not unexpectedly the estimated percentage mortality increases in Table L are typically 40-50% the size of those given in Table XLVI. Reduction by a similar factor occurred upon stratifying on cigarette smoking and hypertension status in the manner described above. All mortality increase estimates were, however, still positive. It seems appropriate to expand a little on some possible disease implications of OC formulations currently consumed relative to those used earlier. Sequential products are no longer in use in most countries, and their influence on risk of endometrial cancer, which was due to their net estrogenic effect on the endometrium, is of little importance today. There has been a marked reduction in the doses of estrogen and progestogen in combined oral contraceptives since the initial products were marketed in the late 1950s and early 1960s. To the extent that this reduction has involved both hormonal constituents about equally, one would expect any adverse or beneficial effects to TABLE L “ESTIMATED” OC-INDUCED PERCENTAGE INCREASEIN CUMULATIVE

hf ORTALITY h T E OVER THE 80-YEAR PERIOD FOLLOWING h”1ATION OF OC USE, AS A FCNCTION OF OC USAGE CHARACTERISTICS FOH A 1 ~ Y I ” T H E T I C h L LOW’ PROCESTOCEN

oc FORMULArIoN

Age at initiation of OC use

Duration of OC use (years)

20

25

30

35

40

2.5 7.5 12.5 17.5

1.1 1.7 2.9 4.5

1.3 2.5 4.8 7.3

1.5 3.1 6.5

1.7 G.7

1.8

1.91

2.75

4.20

6.41

9.78

Estimated 20-year mortality rate (%) among OC never-users

EPIDEMIOLOGY OF ORAL CONTRACEPTIVES AND DISEASE

389

be less for current products than for their predecessors. An exception to this might be effects related to the suppression of ovulation, since this occurs almost universally in users of both modern and older combined oral contraceptives. Thus, if ovulation suppression is the mechanism by which risks of ovarian carcinomas or retention cysts of the ovary have been reduced, then these same benefits should be enjoyed today by users of currently marketed preparations. If, in addition to a reduction in dose, the ratio of estrogen to progestogen in the newer (or future) oral contraceptives is altered, then such formulations might have a net estrogenic or progestogenic effect on target organs that are not necessarily smaller than those of the older preparations. If, for example, some newer pills had more of a progestogenic effect and less of an estrogenic effect than the older preparations, then the reduced risks of benign breast diseases and endometrial carcinoma that have been related to the progestogenic effect of the older preparations might also be observed in users of such newer products in spite of their lower dose. On the other hand, a net progestogenic effect may have adverse implications for major arterial diseases in view of an apparent role of progestogens in blood pressure elevation, and particularly for MI in view of the apparent adverse effects on serum lipids and lipoproteins of high progestogen-low estrogen preparations. To the extent that current preparations reflect reduced progestogen dosages at a given estrogen dosage it is natural to expect corresponding reduced cardiovascular disease risk relative to earlier preparations. There are additional complications toward trying to predict the disease effects of current preparations. For example, triphasic oral contraceptives, with varying doses of estrogen and progestogen during a cycle of use, have been marketed in recent years, and their effects on risks of disease cannot readily be anticipated by epidemiologic studies to date. Furthermore, many progestogenic agents also have other hormonal effects. For example, norethynodrel, norethisterone, ethynodiol diacetate, and lynestrenol are also estrogenic; dl-norgestrel and norethisterone are androgenic; and chlormadinone acetate and cyproterone acetate are anti-androgenic. The influence of specific progestogens in oral contraceptives on risks of various conditions has not been adequately studied. However, qualitative changes over time in the progestogens used in oral contraceptives could alter their effects on risk of various neoplasms, cardiovascular diseases, and other conditions. These considerations indicate that the exercise of Table L represents a quite crude attempt to extrapolate the results of previous epi-

390

ROSS L. PRENTICE AND DAVID B. THOMAS

demiologic studies to current OC formulations. An adequate assessment of the risks and benefits of current formulations must await additional years of follow-up of women using such preparations and an analysis of their corresponding disease experience. An additional qualification of the preceding analyses and tables arises from the fact that the estimated mortality increases pertain only to the 2O-year period following initiation of OC use. The mortality impacts of OCs over a longer period of time since first use may be greater than is suggested b y Tables XLVI-L if a noteworthy relative risk of stroke or myocardial infarction among former OC users persists for a decade or so following cessation of use. Specificially myocardial infarction mortality rates increase very rapidly after age 50, much more so than do mortality rates for diseases for which OC use may convey some benefit. On the other hand, some of the entries in Tables XLVI-L may overestimate long-term mortality risks if alterations in risk among former users persist for only a few years. It is also worth reiterating that the above calculations are intended to be pertinent to U.S. mortality rates. The mortality implications of OC use in developing countries, for example, may be very different (e.g., Belsey, 1982), in particular maternal mortality rates may be very much higher than in the United States. As a very crude indication of the possible implications of an elevated maternal mortality, the riskbenefit mortality calculations of Table XLVI were repeated while multiplying by five the age-specific mortality rates for pregnancy, childbirth, and the puerperium given in Table XLIV. For example, five is approximately the ratio of 1975 maternal mortality rates (per woman) in Thailand compared to those in the United States. Table LI shows that such a change brings about some reductions in estimated increases in OC-associated mortality (compared to Table XLVI) particularly among younger women. The estimated increases in total mortality, however, all remain positive, as also turns out to be the case when cigarette smoking and hypertension are also accommodated. Of course an adequate examination of OC-associated mortality in a specific developing country would need to take account of disease-specific mortality rates, OC-associated relative risks, and contraceptive practices for that country. As mentioned previously the maternal mortality relative risk, taken to be 0.2 in Table XLIII, involves a comparison of current OC users to nonusers having the mixture of contraceptive practices prevailing in the United States (in 1975). Maternal mortality relative risks for current OC use versus specific contraceptive practices or compared to no fertility control will be substantially different (e.g., Jain, 1977). For

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TABLE LI “ESTIMATED” OC-INDUCED PERCENTAGE INCREASE IN CUMULATIVE MORTALITYOVER THE %)-YEAR PERIOD FOLLOWING INITIATION OF oc U SE , AS A FUNCTION OF OC USAGE CHARACTERISTICS, FOR A HYPOTHETICAL POPULATION WITH MATERNAL MORTALITYRATES FIVE TIMES THATOF CORRESPONDING U.S. RATES Age at initiation of OC use

Duration of OC use (years)

20

2.5 7.5 12.5 17.5

1.4 1.8 3.7 6.4

Estimated 20-year mortality rate (%) among OC never-users

1.99

25 2.0 4.0 8.7 14.1 2.79

30

35

40

2.8 6.1 13.8

3.2 7.8

3.7

4.20

6.39

9.71

example, the maternal mortality relative risk for OC users versus intrauterine device (IUD) users appears to be in the vicinity of 0.4 (Ory et al., 1983), rather than 0.2, so that I U D users can be expected to have a slightly greater mortality benefit compared to OC users than is indicated by Tables XLVI-L, assuming I U D use is not itself associated with appreciable mortality. On the other hand, the maternal mortality relative risk for OC use versus a dependence on “rhythm” appears to be in the vicinity of 0.08 so that lesser mortality benefit compared to OC use corresponds. Even a maternal mortality relative risk of zero among current OC users, however, does not substantially affect the calculations in Tables XLVI-L. Let us conclude our risk-benefit comments by recalling that the above calculations are restricted to mortality while, as has been discussed in previous sections, OC use appears to convey important risks and benefits for conditions or diseases that need not be fatal or even involve a mortality risk. Let us turn now to a reiteration of some of the needs for further research into epidemiologic aspects of OC use. As preceding paragraphs implied, one of the most pressing needs is for better information on the risks and benefits associated with current OC formulations. Another important need for both current and former preparations concerns the disease implications of former OC use. Specifically, further studies are needed to assess risks long after cessation of OC use for hemorrhagic and thrombotic stroke, myocardial infarction, invasive cervical carcinoma, hepatocellular carcinoma, and cutaneous malig-

392

ROSS L. PRENTICE AND DAVID B. THOMAS

nant melanoma, and studies are needed to determine the duration and magnitude of protective effects against endometrial and ovarian cancer. Studies of the dependence of these risks and benefits, particularly for myocardial infarction and endometrial cancer, on the subsequent use and formulation of menopausal steroids is also important indeed. Studies of the dependence of disease risk on duration of oral contraceptive use also deserve high priority. Of particular importance are relationships of duration of use to enhanced risks of myocardial infarction, invasive cervical cancer, and liver cancer, and to reduced risks of endometrial and ovarian carcinomas, and possibly also of breast cancer. Subsequent studies of cervical cancer should include assessments of the potential confounding influences of smoking, sexual behavior and the male partners, and serologic indicators of sexually transmitted d'isease. Studies of the dependence of disease risks on potencies of estrogens and progestogens in the oral contraceptives are likewise important toward predicting likely adverse and beneficial effects of currently used preparations, and additionally may help to clarify our understanding of disease mechanisms. In particular, no study appears to have evaluated estrogen and progestogen dosage in relation to possible OC-associated risks of cardiovascular disease among former users. Also, the possible influence on risk of breast cancer of preparations of varying progestogen potency when used early in the reproductive years requires clarification. In addition, the formulations that provide the maximum protection against endometrial cancer should be elucidated. Existing databases, particularly those from the major cohort studies, should be able to help clarify some of these issues, in spite of the difficulty of comparing estrogen potencies and progestogen potencies among marketed OC formulations. Other issues will require additional case-control studies. The relative risk regression methods outlined in Section I1 are well suited to analyses that simultaneously permit OC-associated relative risks to depend on such features as duration of current and former use and estrogen and progestogen potencies in relation to current or former use, attained age, and other characteristics. Further analysis of existing data, particularly from the major cohort studies, using such methods is advocated. The possibility of combining the raw data from two or more such cohorts (as strata) in such an analysis also seems to have merit. In terms of the methods used in this article, let us repeat a comment from Section I1 that the statistical methods used to approximate confidence intervals for the relative risks throughout Sections 111 to V are somewhat crude. We are confident that these approxima-

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tions are not misleading in respect to the summary relative risk estimates and approximate confidence intervals presented, particularly since such summary calculations tend to be dominated by the larger studies for which confidence intervals were often given in the publications cited. Outside of this context, however, the use of such confidence interval approximations should not be regarded as a substitute for well-established confidence interval procedures for the types of studies reviewed here. As a concluding statement let us indicate that much has yet to be learned concerning the relationship between oral contraceptives and disease. Our risk-benefit summary suggests that, at least for OC formulations common in the 1960s and early 1970s, the mortality risks appear to exceed the benefits among most U.S. women. Longer term follow-up and additional study will be required to determine whether such a statement pertains to recent OC formulations.

ACKNOWLEDGMENTS This work was supported by the following grants and contracts: GM-24472, CA15704, HFU181/467, and HD-52901. The authors thank Mark Mason and Elizabeth Noonan for computational assistance. They also thank Drs. Bruce Armstrong, Clifford Kay, Martin Vessey, and others for providing helpful comments on an earlier version of this review.

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INDEX A Abelson murine leukemia virus (AMuLV) isolation from Moloney-MuLV-infected mouse, 53 v-abl oncogene, 54; see also Oncogenes, v-abl Acetylcholine receptors (AChR) in myotubes, RSV ts mutant effect, 11 Acute lymphocytic leukemia (ALL) ambiguous cases, explanation, 139140 classification, 139 relapses after bone marrow transplantation, 156 surface marker assay G6PD phenotype and, 140-141 Ig heavy chain and, 140-141 sensitivity, 141 Acute nonlymphocytic leukemia (ANLL) clonal development, 153-154 G6PD assay, 153-156 multistep pathogenesis, 155-156 relapses after bone marrow transplantation, 156-157 remission, cell heterogeneity and, 154-155 stem cell differentiation, 154 ACV, set. Avian carcinoma virus Adipocytes, differentiation induction by TPA-type tumor promoters, 246 AEV, see Avian erythroblastosis virus African Burkitt’s lymphoma, see Burkitt’s lymphoma Agnogenic myeloid metaplasia, 152-153 AIDS, NK cells defective in NKCF release, 183-184 improvement by IL-2, 184 403

Alkaline phosphatase in FBJ virus-induced osteosarcoma, murine, 30 ALL, see Acute lymphocytic leukemia Allergy, OC effects, 372 3-Aminobenzamide, inhibition of cell malignant transformation, 276, 278 chymotrypsin and trypsin, 216-277 PMA-induced superoxide anion formation by neutrophils, 277 poly(ADP-ribose) synthesis, 276, 278 A-MuLV, see Abelson murine leukemia virus AMV, see Avian myelocytomatosis virus ANLL, see Acute nonlymphocytic Ieukemia Antibodies to EBV antigens during chronic asymptomatic infection, 107 during primary infection, 96 limited role in infection control, 98 monoclonal, see Monoclonal antibodies

Antigens CEA (carcinoembryonic) epitopes, detection by MAbs, 198199 secretion in cell culture, 198 in sera of cancer patients, 209-210 EBV-induced in B cells antibody induction during chronic asymptomatic infection, 107 primary infection, 96-97 latent infection EBNA 1 , 2 , 3 , nuclear, 80,96-97 LIMP, membrane protein, 80, 90, 110

404

INDEX

nonproductive infection EBNA, nuclear, 80 LYDMA, cell surface, 80-81, 109-

110 productive infection EA, early, 81-82 MA, membrane, 82,96 K A , viral capsid, 82 GICA (gastrointestinal cancer) carbohydrate determinants, 196 detection by DDIA in patients’ sera, 207-209 in spent media of tumor cells,

196- 197 in saliva of Lewis-positive individuals, 205-206 PA (prostate) release by tumor cells i t ] oitro and i n cico, 212 in sera of cancer patients, 212-213 PCAA (pancreatic cancer-associated), secretion in tiitro, 199 POA (pancreatic oncofetal), secretion in citro, 199 sialylated X shedding by tumor cells in tiitro,

197-198 in sera of colorectal carcinoma patients, 209 T8 in T-cell malignancy, 142-143 TAA, see Tumor-associated antigens TAG 72, in sera of colorectal carcinoma patients, 210 X, Y, H complex carbohydrate determinants, 197-198 secretion by tumor cells in citro,

197-198 Antipain c-myc expression inhibition, 267, 279 poly-ADP-ribosylation inhibition, 278 Aplysiatoxin biological activities, 226, 238, 241 derivatives, structure-function assay,

228, 239-244 isolation from alga Lyngbyu mujuscula, 227-228 seaweed dermatitis induction, 227-

228 structure, 225, 238-239 tumor-promoting activity, 226-228,

241-244

Arachidonic acid metaboIism, stimulation by palytoxin, 255 TPA-type tumor promoters, 248 Avian carcinoma virus (ACV), SKV 770 strain generation in uitro, 15 myoblast transformation, 16 myogenesis induction in uncharacterized embryonal cells, 15-16 Avian erythroblastosis virus (AEV), myoblast transformation, 12-13 t s mutants and, 13 Avian myelocytomatosis virus (AMV), MC29 strain, myoblast transformation td 10H mutant effect, 15 desmin low level, 14-15 differentiation suppression, 14

B B-cell growth factor, in EBV-activated B cells, 93-94 B cells EBV infection cell surface receptor complement, type 2 (CR2), 83 non-CR2 in VDS-O line, 83-84 chronic asymptomatic fibroblast effects in vitro, 114 low infection frequency, 105-106 oropharynx infection effect, 105-

107 regulatory control of, 106-107 latent, antigens, 80, 90, 110 nonproductive, antigens, 80, 109-

110 primary, EBNA expression, 96-97 productive, antigens, 81-82 T-cell effects during acute IM,98-105 during chronic asymptomatic infection, 107-115 viral DNA integration, 79-80 immortalization by EBV EBNA expression and, 85-87 enhancement by growth factor, 93-94 monocytes, 91-93

405

INDEX

Ig secretion and, 85-87,88-90 mechanisms of, 88, 94-95 proliferation and, 85-86, 88, 90 quantitation, 85-88, 90 serological identification, 90 Benzamide, inhibition of cell malignant transformation, 276, 278 chymotrypsin and trypsin, 276-277 PMA-induced superoxide anion formation by neutrophils, 277 poly(ADP-ribose) synthesis, 276,278 Blood group determinants Lewis” and Lewisb, GICA-associated in saliva of cancer patients, 205-206 in sera of cancer patients, 207 in tumor cell culture, 196-197 Blood pressure, OC effects, 333-335 Bloom’s syndrome, chromosome abnormalities, 273 in vitro reduction by protease inhibitors, 273 B-lymphoid cells clonal evolution, cell markers, 128 Burkitt’s lymphoma and, 138 CLL and, 135-137 EBV-induced in immunosuppressed patients, 138-139 hairy cell leukemia and, 138 heavy chain disease and, 137-138 multiple myeloma and, 135 in CML, differentiation from bone marrow stem cells, 146-147 Ig gene rearrangement, 128-129 Bone marrow stem cell multipotent differentiation into adherent cells, 159 in ANLL, 154 in CML, 145-148 transplantation Ig RFLP differences between host and donor, 157 leukemia relapse cases ANLL, 156-157 CML, 157 stromal environment and, 158 Breast benign diseases, protection by OC, 370-371 Breast cancer OC use effect, 337-343

TAA in patients’ sera, 206 shed in culture, 200 Burkitt’s lymphoma, human EBV detection, 75, 76-77 EBV-induced, clonal development, 138

C Calcium ion, requirement for NK CMC, 173- 174 Carcinogenesis high-protein diet and, 271 metastatic process, block by protease inhibitors, 267 OC use effects, 335-369; see also specijic cancers mortality rate, 297 age-specific, 378-379 relative risk, 375-378 prevention by protease inhibitors in oitro, 267,272-273,278-279 in uivo, 266-272 oxygen radical role, 274-278 protease inhibitor destruction and, 266 Catalase neutrophil DNA protection from PMAinduced damage, 274 PMA-induced inhibition in skin, mouse, 276 Cell cycle, Go-GI transition induction of c-fos and c-myc, 44-45 Cervical cancer, OC use effect, 347-359 Cholesterol, OC effects, 333 Chondroblasts, transformation in vitro, avian by AMV, differentiation block, 21 by RSV differentiation block, 19 specific gene transcription and, 20 Chromosomes abnormalities, hematopoietic neoplasm clonality and, 133-134 in CML hematopoietic cells, 55 Philadelphia (Ph’), 53, 54-55, 5758,70 various rearrangements, 70 X, inactivation mosaicism in hematopoietic neoplasms

406

INDEX

G6PD phenotype and, 130-132 RFLP and, 132-133 Chronic lymphocytic leukemia (CLL) B-cell progenitor clonal disorder G6PD phenotype and, 135 Ig gene rearrangement and, 136-137 Ig light chains and, 135-136 therapy with anti-tumor-cell-idiotype MAbs, 136-137 Chronic myelocytic leukemia (CML) bone marrow stem cell in cjitro differentiation into B-lymphoid cells, 146-147 granulocytes, 145-146 T cells, 147-148 chemotherapy effect, 151 clonal development, assay with GGPD, 144-148 6-phosphogluconate dehydrogenase, 145 future research, 70-71 multistep pathogenesis bcr rearrangements, 150 blast crisis, 55, 150 GGPD assay, 148- 149 Ig gene rearrangement, 150 Ph'-negative stage in Phl-positive B cells, 149 chronic phase, 55, 149 novel c-abl mRNA and related protein, 55 Ph' chromosome in hematopoietic cells, 53, 54-55, 57-58, 70, 144151 Phl-negative, 144, 151-152 bcr rearrangements, 152 relapses after bone marrow transplantation, 157 Chronic myelogenous leukemia, see Chronic myelocytic leukemia Chronic myeloproliferative disorders CML, see Chronic myelocytic leukemia diversity, 152 G6PD phenotype assays, 152-153 CIC, see Immune complexes, circulating Circulatory system diseases, OC effects cerebrovascular disease, 300-312 cigarette smoking and, 310-312 fatal subarachnoid hemorrhage, 303, 305-306

hemorrhagic stroke, 303-305 thrombotic stroke, 303, 306-307 ischemic heart disease, 312-323 myocardial infarction, 3 13-323 mechanisms blood pressure increase, 333-335 glucose tolerance decrease, 334 HDL cholesterol reduction, 333 mortality rate, 297 age-specific, 378-379 peripheral vascular disease, 324-332 pulmonary thromboembolism, 325329 venous thromboembolism, 324-328, 330-332 CLL, see Chronic lymphocytic leukemia CML, see Chronic myelocytic leukemia Collagen type I in FBJ virus-induced osteosarcoma, murine, 30 Colorectal carcinoma -specific CIC, 215 TAA in sera, 206 CEA, 209-210 GICA, 207-209 sialylated X antigen, 209 TAG 72,210 Cytolysins or perforins functional comparison with NKCF, 182 Cytotoxic T cells in EBV chronic asymptomatic infection activity toward B-cell antigens LIMP and, 110 LYDMA and, 109-110 clones, generation and properties, 110-111 HLA restriction, 109, 110-111 virus-nonspecific activity, 114-115 n-interferon role, 115 in EBV-induced acute IM, activation, 99-100

D DDIA, see Double-determinant immunoassay Debromoapl ysia toxin biological activities, 226, 238, 241

407

INDEX

isolation from alga Lyngbia majuscula, 227-228 seaweed dermatitis induction, 227228 tumor-promoting activity, 226-228, 241-244 Des-0-methylolivoretiin B form, identity with teleocidin B-1, 231,233 biological activities, 233 (table) C form, isolation from Streptomyces mediocidicus, 233 structure, 231 comparison with (-)indolactam-V, 234 Differentiation block by oncogenes, 16-17 proliferation-independent, by src, 18-21 via uncontrolled cell proliferation by myc, 21-22 c-fos-induced, gene expression, 46-47 -proliferation relationship, 17-18 -transformation relationship, 22-25 Diet high-protein, contribution to carcinogenesis, human, 271 protease inhibitor-rich human cancer prevention and, 267268 rodent cancer prevention leupeptin and, 269-270 raw soybeans and, 270-271 synthetic compounds and, 271272 Dihydroteleocidin B skin irritant from Streptomyces mediocidicus, 224 structure, 225 tumor-promoting activity, 224-226 7,12-Dimethylbenz[a]anthracene (DMBA) skin cancer induction, mouse, 235,237 aplysiatoxin-promoted, 241-244 teleocidin-promoted, 226-227, 230, 233,235-237 DMBA, see 7,12-Dimethylbenz[a]anthracene DNA amplification carcinogen-induced of SV40, in-

hibition by protease inhibitors, 278 initiator-induced, block by protease inhibitors, 278 complementary (cDNA) to CMLspecific mRNA bcr-abl5’ nucleotide sequences, 62-69 cloning, 58-62 inverted repeat, 62 5’noncoding sequences, 60,62 screening strategy with v-abl probe, 59-60 EBV integration into cellular DNA, 79-80 organization, 77-79 internal repeats, 78-79 polymorphism, 79 PMA-induced strand breaks in neutrophils, 274-275 comparison with y-radiation effect, 275 protection by catalase and SOD, 274 TPA-type tumor promoter effects, 246-247 Double-determinant immunoassay (DDIA) GICA in tumor cell media, 196-197 TAA in patients’ sera, 203-204 colorectal carcinoma and, 207-209

E EBV, see Epstein-Barr virus EGF, see Epidermal growth factor EGF receptor, phosphorylation protein kinase C activation by TPAtype tumor promoters and, 252 Emphysema, cq-trypsin inhibitor in prevention of, 266 Endometrial cancer, OC use effect, 335337 Epidermal growth factor (EGF) c-fos induction, 41,43-45 Epstein-Barr virus (EBV) in B cells, see also B cells antigens, 79-82,90, 109-110 cell immortalization induction, 8495 latent form, 79 receptor nature, 82-84

408

INDEX

chronic asymptomatic infection antibodies to VCA, EBNA, and MA, 107 B-cell infection, 105-107 T-cell effects, 107-115 detection in human cancer African Burkitt’s lymphoma, 75, 7677 nasopharyngeal carcinoma, 75, 77 DNA integration into cellular DNA, 79-80 organization, 77-79 polymorphism, 79 envelope proteins, 82 genes LTI, LT2, LT3, LT4,80-81 IM induction acute, 75, 96-98 cellular immunity during, 98-105 chronic, 76 neoplastic B-cell proliferation induction Burkitt’s lymphoma and, 138 in immunosuppressed patients, 138139 primary infection, 75, 95-98 antibodies to EBV antigens, 96, 98 sites of B cells in peripheral blood, 96-98 oropharynx, 95 Erythroid cells, differentiation block by erbA plus erbB, 17 Essential thrombocythemia, 152- 153 Estrogen, in combined OC, 285-286; see also Oral contraceptives

FBR virus defective FBR-MSV, 33 oncogene product, 33: see ulso Gene products helper, B-tropic, FBR-MLV, 33 isolation from YSr-induced osteosarcoma, murine, 33 Fibroblasts, avian v-myc-transformed, lack of tumorigenicity, 24 v-src-transformed differentiation block, 19 specific gene expression, 20 tumorigenic in nude mice, 24 Fujinami sarcoma virus (FSV) myoblast transformation, 12

ti Gallbladder disease, acceleration by OC, 372-373 Gene products of avian retroviral oncogenes cytoplasmic serine kinase-specific, 7 tyrosine-specific kinases, 6-7 c-a bl-encoded P145’-“*’, human, 54 P150”-n61,murine, 54, 56 c-bcr-abl-encoded P210c-ab’,in CML cells, 57 amino acid sequence, deduction, 62-69 generation from Ph’ chromosome, scheme, 58 hydrophilicity plot, deduction, 63,

70

F FBJ virus bone-associated tumor induction, murine, 30-31 defective FBJ-MSV quantitation, 32 transplantable tumor induction, 32 v-fos oncogene coding for p55,33; see also Oncogenes, v-fos helper, N-tropic, FBJ-MLV, yuantitation, 32 in osteosarcoma extract, murine, 29

properties, 57-58 fos-encoded cellular, synthesis regulation, 48-49 viral and cellular localization in nucleus, 35, 38-39 structure, 33-37 Genes Ig, rearrangements in B-lymphoid cells, 128-129 ALL and, 140-141 CLL and, 136-137 CML and, 150 LTI, 2 , 3 , 4 of EBV, 80-81 procollagen type I, src effects

409

INDIEX

activation in chondroblasts, 20 suppression in fibroblasts, 20 procollagen type 11, src-suppressed in chondroblasts, 20 T-cell receptor, rearrangements in Tcell lymphoid cells, 129-130, 141-143 Glucose-6-phosphate dehydrogenase (G6PD) A and B types, distinction, 130 AIB phenotype as marker for X-chromosome inactivation mosaicism, 130-132 in neoplastic tissues, 130-132 ALL and, 140-141 ANLL and, 153-156 Burkitt’s lymphoma and, 138 CML and, 144-149, 151 various chronic myeloproliferative disorders, 152-153 Glucose tolerance, OC effects, 334 GGPD, see Glucose-6-phosphate dehydrogenase

H Hematopoietic neoplasms, human bone marrow transplantation effects, 156-159 clonality, marker systems cytogenetic abnormalities, 133-134 Ig gene rearrangement, 128-129 T-cell receptor gene rearrangement, 129-130 X-chromosome inactivation mosaicism, female G6PD phenotype and, 130-132 RFLP affecting DNA methylation, 132-133 lymphoproliferative disorders B-cell, 134-141 T-cell, 141-144 rnyeloproliferative disorders, 144-156 Hematopoiesis c-fos induction during, 45-46 neoplasia, see Hematopoietic neoplasms HL-60 cells (human promyelocytic leukemia)

TPA-type tumor promoter effects adhesion induction, 224,226, 230, 233, 236, 241 (tables) differentiation induction, 246 unaffected by non-TPA-type tumor promoters, 254-256 Human T-cell leukemia virus (HTLV) T-cell lymphoproliferative syndrome induction, 143-144

I Ig, see Immunoglobulins IL-2, see Interleukin 2 IM,see Infectious mononucleosis Immune complexes, circulating (CIC) TAA-containing in cancer patients’ sera, 214-215 Immunoglobulins (IG) EBV-induced secretion by B cells cell maturation and, 90 inhibition by suppressor T cells in acute IM, 100-103 in chronic asymptomatic infection, 113-1 14 a-interferon role, 113 single isotype, mostly IgM, 88-89 heavy chain abnormalities ALL and, 140-141 chronic B-cell disorder and, 137 hairy cell leukemia and, 138 light chains K and A in B cells CLL and, 135-137 NHL and, 135 normal, 128 (-)Indolactam-V biological activities, 236 (table) structure, 234 as teleocidin biosynthetic intermediate, 234-235 Infectious mononucleosis (IM), acute, EBV-induced during primary infection, 75-76, 9698 T-cell responses contribution to disease, 104-105 cytotoxic, 99-100 suppressive, 100-103

410

INDEX

a-Interferon TAA shedding in oitro and, 194 T-cell effect on EBV-infected B cells and, 115 y-Interferon TAA shedding in vitro and, 194 T-cell effect on EBV-infected B cells and, 113 Interleukin 2 (IL-2) pretreatment of NK cells from AIDS patients, 184 NKCF secretion induction and, 184

M MAbs, see Monoclonal antibodies az-Macroglobulin inhibitor destroyed in neoplastic lesions, 266 Mammary carcinoma, see Breast cancer Mannose, suppressor T-cell inhibition in

IM, loo, 102 Melanoma antigens shedded in vitro gangliosides, 201, 214 protein, 201 TAA during tumor progression, 192-

193

L Large granular lymphocytes, human NKCF in vitro secretion, 171-172 Leucine as leupeptin metabolite, 269-270 tumor growth enhancement, rodents,

270 Leupeptin dietary, cancer prevention, rodents,

269-270 leucine release as disadvantage,

270 poly-ADP-ribosylation inhibition, 278 Liver cancer, OC use effect, 359-366 Lung carcinoma sialylated X antigen elevation in sera,

213 Lymphotoxin properties, 180 recombinant, comparison with NKCF action, 181 Lyngbya mujuscula (blue-green alga) seaweed dermatitis induction, 227-

228 tumor promoter isolation aplysiatoxin, 227-228 debromoaplysiatoxin, 227-228 lyngbyatoxin A, 227,229 Lyngbyatoxin A identification with teleocidin A-1, 229 isolation from alga Lyngbya majuscula, 227, 229 purification, 229 structure, 226 tumor-promoting activity, 226-227

malignant, OC use effect, 366-369 TAA in sera, 213 CIC and, 214-215 Microfilaments, arrangement in fibroblasts TPA-type tumor promoter effects, 248 Migraine headache, OC effect, 373 Mitogenesis, induction by TPA-type tumor promoters, 247 Monensin, NKCF secretion inhibition,

175 Monoclonal antibodies (.MAbs) anti-tumor-cell-idiotype, CLL therapy,

136-137 in CIC of melanoma patients, 214-

215 TAA detection, see also specijic antigens in sera of cancer patients, 202-215 in tumor cell spent media, 190-201 Monocytes, Ig secretion stimulation in EBV-activated B cells, 91-93 Mucin, high-molecular weight, see Tumor-associated antigens, in vitro shedding Multiple myeloma antibodies, usually class IgC, 137 B-cell clonal proliferation, 135 Ig-secreting plasma cell increase, 134135 Mutants td (transformation-defective) 10H of AMV strain MC29 characteristics, 15 revertant myotube induction, 15 slow myoblast transformation, 15 t s (temperature-sensitive)

INDEX

AEV, myoblast transformation, 13 RSV, myoblast transformation, 4, 10-12 Myoblasts, transformation in oitro quail by ACV strain SKV770, differentiation pattern, 16 by AEV, differentiation pattern, 12-13 revertant myotubes and, 13 t s mutants and, 13 by AMV strain MC29, differentiation pattern, 13-15,21-22 td 10H mutant and, 15 by FSV, differentiation pattern, 12 by RSV differentiation block, 8, 18-21 macroscopic abnormalities, t s mutant-induced, 4, 10-12 revertant myotube formation, 4, 910 spontaneous differentiation and, 4, 8-10 M yogenesis retroviral oncogene effects, see Myoblasts, Oncogenes stages from myoblasts to myofibers, 25 Myosin, in revertant myotubes, distribution, 4 Myotubes, revertant induction by AEV, 13 AMV td 10H mutant, 15 RSV, 9-10 myosin distribution, 4

N Nasopharyngeal carcinoma, EBV detection, 75, 77 Natural killer cell-mediated cytotoxicity (NK CMC) defective in AIDS patients, 183-184 IL-2 stirnulatory effect, 184 description, 169-170 inhibition by monensin and verapamil, 175 kinetics, 176 multistage mechanism, 170-171 NKCF a; soluble mediator, 171-172,

411

176; see also Natural killer cytotoxic factor Natural killer cells (NK cells) NKCF secretion, 170-172; see also Natural killer cytotoxic factor target cell recognition and binding, 172- 173 Ca2+role, 173 Natural killer cytotoxic factor (NKCF) binding by target cells, 176-178 postbinding events, 178 biochemical characterization, 180 functional comparison with cytolysins or perforins, 182 lymphotoxin, 181 TNF, 181-182 liposome-encapsulated, effect on NKresistant cells, 179 secretion by NK cells, 170-172 activation, 173-175 cytosolic Ca2+ and, 174 protein kinase C and, 174 serine-dependent proteases and, 174- 175 inhibition by monensin and verapamil, 175 kinetics, 175-176 large granular lymphocytes, human, 171-172 splenocytes, murine, 171 Nerve growth factor (NGF) c-fos induction in neurites, 46 Neurites c-fos induction during differentiation, 46 protein kinase C and, 46 Neutrophils, human oxidative burst, stimulation by TPAtype tumor promoters, 247-248 PMA-induced HzOe formation, 273 DNA damage and, 274-275 protection by catalase, 274 inhibition by protease inhibitors, 273-274,277 NGF, see Nerve growth factor NHL, see Non-Hodgkin’s lymphoma Nicotinamide, inhibition of chymotrypsin and trypsin, 276-277 PMA-induced superoxide anion formation by neutrophils, 277 poly(ADP:ribose) synthesis, 276

412

INDEX

NK cells, see Natural killer cells NKCF, see Natural killer cytotoxic factor NK CMC, see Natural killer cell-mediated cytotoxicity Non-Hodgkin's lymphoma (NHL) progenitor 8-cell clonal disorder, 135

0 OC, see Oral contraceptives ODC, see Omithine decarboxylase Olivoretins forms A, B, C, and I) separation, 232-233 structure, 232-233 D form identity with teleocidin B4,231,233 isolation from Streptoverticillium blastomycetum, 234 S. olivoreticuli, 232 Oncogenes avian retroviral products of, 6-8 properties, from various viruses, 7 (table) protooncogene conversion to, 6 transformation induction in tiitro, 45 t s and trl mutants used for, 5-6 c-ctbl comparison with v-abt, murine, 54 expression, 54-57 novel in Ph'-positive CML cells, human, 55 organization, 55-56 c-bcr, rearrangements in CML Phi-negative cells, 152 Ph'-positive cells, 150 c-bcr-obE, expression in CML cells, 57-70; see also Gene products; DN4 c-fos induction cell growth and, 42-45 c-fos-induced differentiation and, 46-47 hematopoiesis and, 45-46

neuronal differentiation and, 46 prenatal development and, 42-43 in regenerating liver, 45 in various cell types, 41 (table) organization, 33-37 recombinants with v-fos construction, 39-40 transforming potential, 39-41 transcription, sequences essential for, 48-49 c-rnyc expression inhibition by antipain, 267,279 c-ras, hansfection suppression b y protease inhibitors, 267 TPA-type tumor promoter effects, 252-253 v-abl of A-MuLV comparison with c-abl, 54 tyrosine kinase activity, 54 v-erb of AEV, myoblast transformation differentiation block, 12 revertant myotubes and, 13 t s mutant effects, 13 v-erbA plus v-erb3 of AEV erythroid cell differentiation black, 17 v-fos of FBJ and FBR cellular transformation induction, 37, 39 in recombinations with c-fos,3941 organization, 33-37 v-fps of FSV, myoblast spontaneous differentiation and, 12 v-myc of AMV, myoblast transformation differentiation block, 13-15 via uncontrolled proliferation, 2122 td 10H mutant-induced revertant myotubes, 15 v-ski of ACV myoblast transformation, 16 myogenesis induction in uncharacterized embryonal cells, 15-16 v-src of RSV chrondroblast transformation differentiation block, 19 specific gene transcription and, 20

INDEX

fibroblast transformation differentiation block, 19 specific gene expression and, 20 tumorigenicity and, 24 myoblast transformation differentiation block, 8, 18-21 specific gene transcription and, 19-21 spontaneous differentiation, 8-12 t s mutant effects, 10-12 Oral contraceptives (OC) allergic diseases and, 372 cancer risk, 375-378 breast, 337-343 cervical, 347-359 endometrial, 335-337 liver, 359-366 malignant melanoma, 366-369 mortality rate, 297 age-specific, 378-379 ovarian, 343-347 cerebrovascular disease and, 300-312 relative risks, current and former users, 300-307,375-378 dependence on dose and duration of use, 307-312 circulatory system diseases mechanisms of, 332-335 mortality rate, 297 age-specific, 378-379 relative risk, 375-378 future research, 391-393 gallbladder disease acceleration, 372373 ischemic heart disease and, 312-323 myocardial infarction, 313, 315, 320321 relative risk, current and former users, 312-318 dependence on dose and duration of use, 319-323 migraine headaches and, 373 mortality relative risks, 295-300, 381388,390-391 peripheral vascular disease and, 324332 relative risk, current and former users, 324-330 dependence on dose and duration of use, 330-332

413

protection against benign breast diseases, 370-371 ovarian retention cysts, 371 rheumatoid arthritis and some thyroid diseases, 372 study methods, 287-295 case-control studies, 290-292 cohort studies, 289-290 consistency among studies, 292-295 relative risk estimation, 288-289 usage age-dependent, estimated frequencies, 378, 380 combined (estrogen + progestogen), 285-286 minipill (progestogen), 286 Ornithine decarboxylase (ODC) induction by TPA-type tumor promoters, 224, 226, 230, 233, 236, 241 O y z i a s Zatipes (teleost), teleocidin toxicity toward, 224 Osteosarcoma, murine FBJ-MSV-induced, transplantable, 32 FBJ virus-induced alkaline phosphatase activity, 30 collagen type I production, 30 lack of metastases, 31 Ovarian carcinoma OC use effect, 343-347 -specific CIC, 215 TAA shedding in vitro, 199-200 in vivo, 206, 212 Ovarian retention cysts, protection by OC, 371 Oxidative burst, neutrophils and macrophages activation by TPA-type tumor promoters, 247-248 Oxygen radicals cell metabolism and, 275-276 DNA damage induction, 274-275 comparison with y-radiation effect, 275 protection by catalase, 274 PMA-induced in neutrophils, 273 inhibition by protease inhibitors, 273-274,277 poly(ADP-ribose) induction and, 277

414

INDEX

P Palythoa (marine coelenterate) palytoxin isolation from, 224, 254 Palytoxin arachidonic acid metabolism stimulation, 255 isolation from coelenterate Palythoa, 224, 254 non-TPA-type tumor promotion, 255257 mechanism of, 256 strong skin irritant, 254-255 structure, 254 Pancreatic carcinoma TAA in sera, 206 (table) GICA elevation, 210 PDGF, see Platelet-derived growth factor Phorbol-12-myristate-13-acetate (PMA) oxygen radical induction in neutrophils, human, 273 DNA damage and, 274-275 protection by catalase, 274 inhibition by protease inhibitors, 273-274 skin cancer promotion, mouse, 268269 inhibition by protease inhibitors, 269 SOD and catalase inhibition in skin, mouse, 276 6-Phosphogluconate dehydrogenase, CML clonality and, 145 Plasma cells, ig-secreting, in multiple myeloma, 134-135 Platelet-derived growth factor (PDGF), c-fos induction, 41-44 PMA, see Phorbol- 12-myristate-13-acetate Poly(ADP-ribose) functions, 278 isolation from peritoneal macrophages, 276 PMA-induced, 276 properties, 276 synthesis, inhibition by protease inhibitors, 276-278 Polycythemia vera, 152-153 Polymorphonuclear leukocytes, see Neutrophils

Potato inhibitors, suppression of chymotrypsin activity, 273 HzOz formation by neutrophils, 274 Progestogen, in combined OC and minipills, 286; see also Oral contraceptives Prostaglandin Ez, TAA shedding in uitro and, 194 Prostate carcinoma, PA in sera, 212213 Protease inhibitors dietary human cancer prevention, 267-268 rodent cancer-preventing leupeptin, 269-270 in raw soybeans, 270-271 synthetic, 271-272 in uitro inhibitory effects on chromosome abnormalities in Bloom’s syndrome, 273 DNA amplification, 278-279 neoplastic transformation, 267, 272273 PMA-induced poly(ADP4bose) synthesis, 276-278 oxygen radical and, 277 superoxide radical formation by neutrophils, 273-274 metastatic process block, 267 oncogene expression suppression, 267 PMA-promoted skin cancer inhibition, mouse, 269 Protein kinase C activation by TPA-type tumor promoters, 251-252 E G F receptor function and, 252 c-fos induction during neuronal differentiation and, 46 NKCF secretion and, 174 unaffected by non-TPA-type tumor promoters, 256 Protein kinases, avian retrovirus-encoded cytoplasmic serine-specific, 7 tyrosine-specific, 6 homologous to growth factor receptors, 6-7 Protein synthesis, induction by TPAtype tumor promoters, 248249

415

INDEX

R Restriction fragment length polymorphism (RFLP) bone marrow transplantation and, 157 cytosine methylation in DNA and, 133 neoplasm clonality recognition, 132133 X-linked, 133 Rheumatoid arthritis, risk reduction by OC, 372 RNA, messenger (mRNA),CML-specific cDNA clones to, see DNA detection, 55, 57 translation to P210c-ab'protein, 58 Rous sarcoma virus (RSV),myoblast transformation in uitro differentiation block, 8 proliferation-independent, 18-21 spontaneous differentiation conditions for, 8-12 revertant myotubes and, 4,9-10 ts mutant-induced, 4, 10-12

S Saliva, GICA and Lewis determinants, human, 205-206 Seaweed dermatitis, Lyngbya majuscula-induced, 227-228 aplysiatoxin and debromoaplysiatoxin role, 227-228 Skin cancer, mouse DMBA-induced aplysiatoxin and its derivativepromoted, 241-244 teleocidin-promoted, 226-227, 230, 233,235-237 inhibition by various compounds, 237-238 PMA-promoted, 268-269 inhibition by protease inhibitors, 269 Skin irritation, mouse ear non-TPA-type tumor promoters and, 254-256 TPA-type tumor promoters and, 224, 226,230,233,236,241 SOD, see Superoxide dismutase

Splenocytes, NKCF in uitro secretion, murine, 171 Stable pancytopenia, 153 Streptomyces mediocidicus des-0-methylolivertin C isolation from, 233 dihydroteleocidin B isolation from, 224 teleocidin isolation from, 227, 228 Streptouerticillium blastomyceticum olivoretins A, B, C, and D isolation from, 234 S. oliuoreticuli olivoretins A, B, C, and D isolation from, 232 Superoxide dismutase (SOD) inhibition by PMA in skin, mouse, 276 neutrophil DNA protection from PMAinduced damage, 274 Superoxide radicals, see Oxygen radicals Suppressor T cells in EBV chronic asymptomatic infection inhibition of Ig secretion by B cells, 111-1 13 y-interferon role, 113 nonspecific effector phase, 114 in EBV-induced acute IM inhibition of Ig secretion by B cells, 100-103 prevention by mannose and its derivatives, 100, 102 lack of cytotoxicity, 103 nonspecific activation, 100-102

T T-cell lymphoproliferative disorders HTLV-induced syndromes, 143-144 T/3 gene rearrangements, 141-143 in mycosis fungoides, 143 in T-cell ALL, 143 T-cell receptor chains a,p, and y, 141 as marker for T-lymphoid cells, 129130, 141-143 T cells in CML, differentiation from bone marrow stem cells, 147-148 cytotoxic, see Cytotoxic T cells

416

INDEX

in EBV chronic asymptomatic infection B-cell immortalization inhibition,

107-109 cytotoxicity, 109-1 11 suppression, 111-114 in EBV-induced acute IM cytotoxicity, 99-100 i g secretion inhibition, 100-103 prevention by mannose and its derivatives, 100, 102 immune response prevention, 104-

105 short life i n citro and in cico, 104 suppressor, see Suppressor T cells T8-positive in T-cell malignancy, 142-

143 Teleocidin (- jindolactam-V as biosynthetic intermediate, 234-235 isolation from Sfreptornyces mediocidicus, 227, 228

mixture of A and B forms, 227 structure, 225,227 toxicity to teleost, 224 tumor-promoting activity comparative to TPA, 226-227, 230,

233,235-237 inhibition by various compounds,

237-238 stronger than by TPA, 249-250 Teleocidin A biological activities, 236 (table) structure, 225 two isomers, 227 A-1, identity with lyngbyatoxin A,

229 biological activities, 230 (table) separation, 229 Teleocidin B conversion to dehydroteleocidin B,

224 four isomers, 227 B-1, identity with des-0-methylolivoretin C, 231, 233 B-4, identity wlth olivoretin D, 231,

233 biological activities, 230 (table) separation, 229 structure, 232 structure, 225

12-0-Tetradecanoylphorbol-13-acetate

(TPA) biological activities, comparison with new tumor promoters, see TPAtype tumor promoters c-fos induction during cell growth, 41-44 during hematopoiesis, 45-46 Thapsigargin isolation from roots of Thupsiu garganicu, 255 non-TPA-type tumor promotion, 255-

257 mechanism of, 256 skin irritation induction, 255 histidine decarboxylase activation and, 256 Theophylline, TAA shedding in vitro and, 194 Thyroid diseases, risk reduction by OC,

372 T-lymphoid cells, T-cell receptor gene rearrangement, 129-130, 141-143 TNF, see Tumor necrosis factor TPA, see 12-O-Tetradecanoylphorbol-13acetate TPA-type tumor promoters aplysiatoxin and its derivatives, 238-

244 biological activities, tests, 224, 226, 230,233,236,241 (tables) discovery, 223-228 mechanism of action oncogene expression and cell transfection changes, 252-253 protein kinase C activation, 251-252 EGF receptor function and, 252 scheme, 256 teleocidin and its derivatives, 228-

238 TPA-specific effects differentiation and, 246 DNA changes, 246-247 intertissue and interspecies variation, 250-251 microfilament arrangement in fibroblasts and, 248 mitogenic stimulation, 247 new protein synthesis induction,

248-249 oxidative burst from neutrophil and

417

INDEX

macrophage stimulation, 247248 virus-induced transformation and, 245-246 al-Trypsin inhibitor destroyed in disseminated neoplastic disease, 266 emphysema prevention and, 266 Tumor-associated antigens (TAA) in CIC of cancer patients’ sera, 214215 in uitro shedding antigen specificity, expression pattern and 191-193 cellular specificity, 190-191 glycolipids, 201 high-molecular weight (HMW), mucin-type, 194-196 CEA, 198-199 GICA, 196-197 from ovarian carcinoma, 199-200 PCAA, 199 POA, 199 X, Y, H complex, 197-198 a- and y-interferon effects, 194 kinetics, 193-194 proteins, 200-201 in uivo shedding blood group and blood group-related antigen in saliva, 205, 206 detection in sera, methods, 203-205 DDIA, 203-204 immunodiagnosis and, 201-202 in sera of patients with bladder and lung carcinoma, 213 colorectal carcinoma, 207-210 mammary carcinoma, 202,2102 12 melanoma, 213

ovarian carcinoma, 212 pancreatic carcinoma, 210 prostate carcinoma, 212-213 Tumor cells, NK-resistant conversion to NK-sensitive cells, 179180 fusion with liposome-encapsulated NKCF and, 179 factors responsible for, 178-179, 183 (table) Tumor necrosis factor (TNF), similarity with NKCF, 181-182 Tumor promoters non-TPA-type, see Palytoxin, Thapsigargin TPA-type, see TPA-type tumor promoters

U Urinary bladder carcinoma, human antigen description, 213 Uterine leiomyoma, human G6PD phenotype as marker, 130-131

V Verapamil, NKCF secretion inhibition, 175

W W-7, teleocidin tumor-promoting activity inhibition, 237 mechanism of, 238

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